Methods for detecting LP-PLA2 activity and inhibition of LP-PLA2 activity

ABSTRACT

This invention relates to methods for determining the activity of Lp-PLA2 in at least one sample from an animal. The invention also relates to methods for determining the inhibition of Lp-PLA2 activity in samples from animals that are administered an Lp-PLA2 inhibitor.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 12/817,677, filed onJun. 17, 2010, now Pub. No. US-2010-0256919-A1, which is a continuationof U.S. Ser. No. 11/106,239, filed on Apr. 14, 2005, now U.S. Pat. No.7,741,020, which claims benefit of U.S. Provisional Application No.60/563,078, filed Apr. 16, 2004, the entirety of which is incorporatedby reference.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

FIELD OF THE INVENTION

This invention relates generally to methods and materials fordetermining lipoprotein-associated phospholipase A2 (herein “Lp-PLA2”)enzyme activity and inhibition of activity in tissue samples fromanimals.

BACKGROUND OF THE INVENTION

Coronary heart disease (herein “CHD”) is the leading cause of death inmany industrial countries. Atherosclerosis is a form of arteriosclerosisor hardening of the arteries in which there is the progressive build-upof plaque containing cholesterol and lipids in blood arteries. Thisbuild-up is associated with an increased risk of heart disease andmorbid coronary events. The build-up of plaque in the arteries isassociated with an immune response that is triggered by damage to theendothelium. Initially, monocyte-derived macrophages accumulate at thedamaged site, due to the immune response causing a migration andaccumulation of smooth muscle cells which form fibrous plaque incombination with the macrophages, lipids, cholesterol, calcium salts andcollagen. The growth of such lesions can eventually block the artery andrestrict blood flow.

Lp-PLA2, also known as PAF acetylhydrolase, is a secreted,calcium-independent member of the growing phospholipase A2 superfamily(Tew, et al. (1996) Arterioscler Thromb Vasc Biol. 16(4):591-9;Tjoelker, et al. (1995) Nature 374(6522):549-53). It is produced bymonocytes, macrophages, and lymphocytes and is found associatedpredominantly with LDL (.about.80%) in human plasma. The enzyme cleavespolar phospholipids, including sn-2 ester of1-O-alkyl-2-scetyl-sn-glycero-3-phosphocholine, otherwise known asplatelet-activating factor (herein “PAF”) (Tjoelker, et al. (1995)Nature 374(6522):549-53).

Many observations have demonstrated a pro-inflammatory activity ofoxidized LDL when compared with native unmodified lipoproteins. One ofthe earliest events in LDL oxidation is the hydrolysis of oxidativelymodified phosphatidylcholine, generating substantial quantities oflysophosphatidylcholine (herein “lyso-PC”) and oxidized fatty acids.This hydrolysis is mediated solely by Lp-PLA2 (i.e., Lp-PLA2 hydrolyzesPAF to give lyso-phosphatidylcholine [herein “lyso-PC”] and acetate).(Stafforini, et al. (1997) J. Biol. Chem. 272, 17895)

Lyso-PC is suspected to be a pro-inflammatory and pro-atherogenicmediator. In addition to being cytotoxic at higher concentrations, it isable to stimulate monocyte and T-lymphocyte chemotaxis, as well asinduce adhesion molecule and inflammatory cytokine expression at moremodest concentrations. Lyso-PC has also been identified as the componentof oxidized LDL that is involved in the antigenicity of LDL, a featurethat may also contribute to the inflammatory nature of atherosclerosis.Moreover, lyso-PC promotes macrophage proliferation and inducesendothelial dysfunction in various arterial beds. The oxidized fattyacids that are liberated together with lyso-PC are also monocytechemoattractants and may also be involved in other biological activitiessuch as cell signaling). Because both of these products of Lp-PLA2hydrolysis are potent chemoattractants for circulating monocytes,Lp-PLA2 is thought to be responsible for the accumulation of cellsloaded with cholesterol ester in the arteries, causing thecharacteristic “fatty streak” associated with the early stages ofatherosclerosis.

Lp-PLA2 has also been found to be enriched in the highly atherogeniclipoprotein subfraction of small dense LDL, which is susceptible tooxidative modification. Moreover, enzyme levels are increased inpatients with hyperlipidaemia, stroke, Type 1 and Type 2 diabetesmellitus, as well as in post-menopausal women. As such, plasma Lp-PLA2levels tend to be elevated in those individuals who are considered to beat risk of developing accelerated atherosclerosis and clinicalcardiovascular events. Thus, inhibition of the Lp-PLA2 enzyme would beexpected to stop the build up of this fatty streak (by inhibition of theformation of lysophosphatidylcholine), and so be useful in the treatmentof atherosclerosis.

Lp-PLA2 inhibitors inhibit LDL oxidation. Lp-PLA2 inhibitors maytherefore have a general application in any disorder that involves lipidperoxidation in conjunction with the enzyme activity, for example inaddition to conditions such as atherosclerosis and diabetes otherconditions such as rheumatoid arthritis, stroke, myocardial infarction(Serebruany, et al. Cardiology. 90(2):127-30 (1998)); reperfusion injuryand acute and chronic inflammation. In addition, Lp-PLA2 is currentlybeing explored as a biomarker of coronary heart disease (Blankenberg, etal. J Lipid Res. 2003 May 1) and arteriosclerosis (Tselepis and Chapman.Atheroscler Suppl. 3(4):57-68 (2002)). Furthermore, Lp-PLA2 has beenshown to play a role in the following disease: respiratory distresssyndrome (Grissom, et al. Crit. Care Med. 31(3):770-5 (2003);immunoglobulin A nephropathy (Yoon, et al. Clin Genet. 62(2):128-34(2002); graft patency of femoropopliteal bypass (Unno, et al. Surgery132(1):66-71 (2002); oral inflammation (McManus and Pinckard. Crit. RevOral Biol Med. 11(2):240-58 (2000)); airway inflammation andhyperreactivity (Henderson, et al. J Immunol. 15; 164(6):3360-7 (2000));HIV and AIDS (Khovidhunkit, et al. Metabolism. 48(12):1524-31 (1999));asthma (Satoh, et al. Am J Respir Crit. Care Med. 159(3):974-9 (1999));juvenile rheumatoid arthritis (Tselepis, et al. Arthritis Rheum.42(2):373-83 (1999)); human middle ear effusions (Tsuji, et al. ORL JOtorhinolaryngol Relat Spec. 60(1):25-9 (1998)); schizophrenia (Bell, etal. Biochem Biophys Res Commun. 29; 241(3):630-59 (1997)); necrotizingenterocolitis development (Muguruma, et al. Adv Exp Med. Biol.407:379-82 (1997)); and ischemic bowel necrosis (Pediatr Res.34(2):237-41 (1993)).

Lp-PLA2 activity from human tissue samples has been measured usingspectrophotometric activity and fluorogenic activity assays (CaymanChemical Company, and Karlan Research Products). See also Kosaka, et al.Clin Chem Acta 296(1-2):151-61 (2000) and Kosaka, et al. Clin Chem Acta312(1-2):179-83 (2001). For instance, Azwell, Inc. (Osaka, Japan)reported in 2000 the synthesis and use of1-myristoyl-2-(p-nitrophenylsuccinyl) phosphatidylcholine as acolorimetric substrate for measurement of human PAF AH (Lp-PLA2)activity in plasma and serum. In 2002, Azwell launched itsresearch-use-only Auto PAF AH assay kit that utilizes this substrate andis formatted for use in a clinical chemistry analyzer. These methods maybe capable of detecting inhibition of Lp-PLA2 activity when an inhibitorof Lp-PLA2 is added to a tissue sample in vitro. However, the methodsprovided with the Auto PAF AH assay are insensitive to measuringinhibition of Lp-PLA2 activity when an inhibitor of Lp-PLA2 has beenadministered to an animal prior to tissue sample collection.

In order to measure Lp-PLA2 activity in the presence of inhibitor in atissue sample obtained from an animal administered inhibitor, anactivity protocol is required. Accordingly, methods for determiningLP-PLA2 activity and inhibition from a tissue sample obtained from ananimal that has been administered an Lp-PLA2 inhibitor are greatlyneeded.

SUMMARY OF THE INVENTION

In one aspect of the present invention, a method is provided fordetermining inhibition of Lp-PLA2 enzyme activity in at least one tissuesample comprising the steps of preparing a solution comprising asubstrate for Lp-PLA2 comprising a colorimetric or fluorometricdetectable moiety; contacting at least one said tissue sample with thesolution of the preparing step; and detecting Lp-PLA2 activity, whereinthe tissue sample is from an animal that has been administered withLp-PLA2 inhibitor.

In another aspect of the current invention, a method is provided fordetermining Lp-PLA2 enzyme activity in a tissue sample obtained from ananimal comprising the steps of:

-   -   a) contacting 110 of a solution comprising:        -   a solution comprising 90 mM            1-myristoyl-2-(4-nitrophenylsuccinyl) phosphatidylcholine            contacted with a solution comprising 200 mM HEPES, 200 mM            NaCl, 5 mM EDTA, 10 mM CHAPS, 10 mM sodium 1-nonanesulfonate            at a pH 7.6 in a ratio of 0.66 μL to 110 μL; with at least            one 25 μL tissue sample from an animal; with 25 μL each of a            p-nitrophenol standard solution comprising; 4, 3, 2, 1, 0.4            or 0.2 nmol/μL p-nitrophenol in methanol; and 25 μL of            phosphate buffered saline (PBS) or ddH₂O to make a blank;            and    -   b) determining Lp-PLA2 activity.

DETAILED DESCRIPTION OF THE INVENTION Glossary

“Animal” as used herein includes any human or non-human mammal, or anyother vertebrate capable of naturally producing an enzyme having Lp-PLA2activity, including Lp-PLA2, Lp-PLA2-homologs or orthologs thereof.

“Clinical trial” means human clinical trial.

“Lp-PLA2 enzyme activity” as used herein includes, but is not limitedto, any enzyme activity of Lp-PLA2. This activity may include but is notlimited to an Lp-PLA2 enzyme binding substrate, releasing product,and/or hydrolyzing phospholipids or other molecules.

“Polypeptide(s)” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. “Polypeptide(s)” refers to both short chains, commonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins. Polypeptides may comprise amino acidsother than the 20 gene encoded amino acids. “Polypeptide(s)” comprisethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may comprise many types of modifications.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino or carboxyl termini.Modifications comprise, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, proteolyticprocessing, phosphorylation, prenylation, racemization, glycosylation,lipid attachment, sulfation, gamma-carboxylation of glutamic acidresidues, hydroxylation and ADP-ribosylation, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins, such asarginylation, and ubiquitination. See, for instance, PROTEINS—STRUCTUREAND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman andCompany, New York (1993) and Wold, F., Posttranslational ProteinModifications: Perspectives and Prospects, pgs. 1-12 inPOSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed.,Academic Press, New York (1983); Seifter et al., Meth. Enzymol.182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

“Filtration” or “filtering” as used herein includes, but is not limitedto, the removal of any substance from a solution and may comprisepassing a solution containing the substance to be removed through filterpaper, Whatman paper, cheese cloth, or a column that selectively removessaid substance from solution based on its physical and/or chemicalcharacteristics. Physical and chemical characteristics that may be usedto remove a substance through filtration may include, but are notlimited to, ionic charge, size, weight, polarity, and/or chemicalmoieties associated with the substance that make it likely to bind tothe material filling the column. Filtration may comprise using gravity,vacuum, and/or centrifugation to facilitate the removal of saidsubstance from solution.

“Scintillation cocktail” as used herein is a mixture of solutes andsolvents, typically containing an organic solvent capable ofsolubilizing and maintaining a uniform suspension of a tissue sample forliquid scintillation. The process of liquid scintillation involves thedetection of beta decay within a sample via capture of beta emissions. Ascintillation cocktail mixture is designed to capture the beta emissionand transform it into a photon emission which can be detected via aphotomultiplier tube within a scintillation counter. Severalscintillation cocktails are commercially available. It is understoodthat a modification of the composition of the scintillation cocktail caneffect and/or optimize the detectable reading from liquid scintillationdepending on the sample.

“Tissue(s)” as used herein comprises serum, cell lysate, tissue lysate,urine, blood plasma, plaque, monocytes, or macrophage cells. Thesetissues can be from humans, non-human mammals or other animals thatnaturally produces and enzyme having Lp-PLA2 activity, includingLp-PLA2, Lp-PLA2-homologs or orthologs thereof.

“Colorimetric or fluorimetric detectable moiety” as used herein is aportion of a compound capable of producing a detectable or measurablesignal. Such a signal may be measurable by, but not limited to, visiblelight emission or absorption, fluorescence, phosphorescence or otherdetectable quanta. For instance, a substrate for Lp-PLA2 may comprise acolorimetric c moiety bonded to phosphatidylcholine at the Lp-PLA2cleavage site. When Lp-PLA2 cleaves the colorimetric moiety fromphosphatidylcholine the colorimetric moiety emits a detectable signal asvisible light. One non-limiting example of phosphatidylcholine bonded toa colorimetric moiety is 1-myristoyl-2-(4-nitrophenylsuccinyl)phosphatidylcholine.

Lp-PLA2 “inhibitor” or “inhibition” as used herein refers to any method,technique, condition, or compound capable of reducing or eliminatingLp-PLA2 activity, including but not limited to reducing or eliminatingany of the activities of Lp-PLA2 including, but not limited to, enzymebinding substrate, releasing product, and/or hydrolyzing phospholipidsor other molecules. Inhibition of Lp-PLA2 activity may be measured in asample obtained from an animal administered an inhibitor, which isconsidered in vivo administration. Alternatively, an inhibitor may beadded to a sample after it is obtained from an animal, which would beconsidered in vitro administration.

As used herein, “reduce” or “reducing” refers to a decrease orelimination in Lp-PLA2 enzyme activity. Some non-limiting examples forthe purposes of measuring reduced Lp-PLA2 activity include measuringLp-PLA2 activity from the same animal in the presence and absence of aninhibitor of Lp-PLA2 activity. Alternatively, Lp-PLA2 activity can bemeasured against a standard recombinantly expressed, semi-purified orpurified enzyme.

As used herein “free” or “essentially free” of Lp-PLA2 inhibitor refersto a tissue sample that contains either no Lp-PLA2 inhibitor or Lp-PLA2inhibitor at a low enough concentration such that Lp-PLA2 activity isnot inhibited by the inhibitor. For instance, if the inhibitor ispresent at a concentration lower than the determined dissociationconstant of that inhibitor for Lp-PLA2, a tissue sample may beconsidered essentially free of inhibitor. A tissue sample may beconsidered free of Lp-PLA2 inhibitor if it is obtained from an animalprior to administration of an Lp-PLA2 inhibitor that is not producednaturally by the animal. A tissue sample may also be considered free oressentially free of an Lp-PLA2 inhibitor if it is obtained from ananimal at a time after the last dose of inhibitor sufficient to ensureclearance based on pharmacokinetic profile of that inhibitor in thespecies of animal.

Lp-PLA2 is a known hydrolyzer of phospholipids. Lp-PLA2 can cleavephospholipids at the sn-2 position to create lyso-PC and oxidized fattyacids. PAF has a two-carbon acyl group at the sn-2 position; therefore,when PAF is hydrolyzed by Lp-PLA₂, the short acyl group is cleaved aswater soluble acetate from the remainder of the molecule, which islyso-PC. A substrate possessing a colorimetric or fluorimetrc moiety canbe used to measure Lp-PLA2 activity. For instance, the substrate,1-myristoyle-2-(p-nitrophenylsuccinyl)-phosphatidylcholine, is a PAFanalogue with a 4-nitrophenyl group conjugated onto a succinyl chain atsn-2 position. Lp-PLA2 (PAF-AH) hydrolyzes the sn-2 position of thesubstrate, producing 4-nitrophenyl succinate. This liberation can bespectrophotometrically monitored at 405 nm and Lp-PLA2 activitydetermined from the change in absorption.

The methods of the present invention have been shown to demonstrate acorrelation between Lp-PLA2 inhibitor concentration in a tissue sampleand Lp-PLA2 activity in vitro. Furthermore, the present inventionprovides methods for measuring Lp-PLA2 activity over time in tissuesamples from animals treated with Lp-PLA2 inhibitor. These data may becorrelated with the pharmacokinetic profile of inhibitor from an animal,such as a human.

A colorimetric Lp-PLA2 activity monitoring assay has been developedusing 1-myristoyl-2-(p-nitrophenylsuccinyl) phosphatidylcholine as thesubstrate. In vitro drug inhibition study using Lp-PLA2-specificinhibitors showed specificity of this substrate against Lp-PLA2.However, the Auto PAF AH assay provided by Azwell failed to detect druginhibition in human subjects who received Lp-PLA2 inhibitor drugs invivo, although the same substrate and the same buffer condition are usedin the assays developed herein. Factors such as pre-incubation of plasmawith assay buffer, plasma sample volume, substrate concentration, anduse of buffer R2A, have been identified to contribute to in vitro drugdissociation in the assay and in turn cause the inability of the assayto detect drug inhibition in in vivo drug-bound tissue samples. Thesefactors therefore were modified in development of new, drug-sensitivecolorimetric Lp-PLA2 activity assays. Interactions between these factorshave also been studied so that assay conditions could be chosen thatwould generate detectable in vivo drug inhibition and also offer anadequate assay dynamic range. This modified drug-sensitive assay is ableto detect 85-95% drug inhibition in human subjects with in vivoadministration of Lp-PLA2 inhibitors and therefore could be used as amonitoring assay to assess drug efficacy in the clinic. This assay alsooffers a dynamic range of close to 100-fold and potentially is alsouseful as a screening assay that is capable of measurement of a broaderrange of Lp-PLA2 activity.

In one aspect of the present invention, a method is provided fordetermining inhibition of Lp-PLA2 enzyme activity in at least one tissuesample comprising the steps of preparing a solution comprising asubstrate for Lp-PLA2 comprising a colorimetric or fluorometricdetectable moiety; contacting at least one said tissue sample with thesolution of the preparing step; and detecting Lp-PLA2 activity, whereinthe tissue sample is from an animal that has been administered withLp-PLA2 inhibitor. These methods may further comprise comparing Lp-PLA2activity from a tissue sample obtained from an animal prior to Lp-PLA2inhibitor administration or that is free of Lp-PLA2 inhibitor.Inhibition of Lp-PLA2 activity may be measured in a plurality of tissuesamples obtained from an animal at more than one time point afteradministration of said Lp-PLA2 inhibitor. The substrate may be1-myristoyl-2-(4-nitrophenylsuccinyl) phosphatidylcholine and may beused at a concentration of about 53 μM to about 1125 μM. Theconcentration of -myristoyl-2-(4-nitrophenylsuccinyl)phosphatidylcholine may be 440 μM or it may be 112 μM.

In one aspect of the invention, the tissue sample may be blood plasma,or it may be serum. In another aspect, the blood plasma is diluted about3 to 9 fold with the solution of the preparing. Lp-PLA2 activity may bemeasured by measuring optical density of the tissue sample.

In another aspect of the present invention, the solution comprising asubstrate for Lp-PLA2 further comprises a buffer and wherein the bufferis incubated with the substrate prior to contacting the substrate withsaid tissue sample. In another aspect, the buffer does not comprisecitric acid monohydrate. In another aspect, the substrate concentrationis maintained at approximately the Km of said substrate. Km of saidsubstrate may be decreased by removing citric acid monohydrate from thebuffer. When the substrate is 1-myristoyl-2-(4-nitrophenylsuccinyl)phosphatidylcholine, the substrate concentration may be about 440 μM ormay be about 112 μM.

In another aspect of the present invention, the volume of plasma sampleis about 15 μL to about 50 μL in a volume of about 125 μl, to about 170μL of the solution of the preparing step. In another aspect, the pH ofthe reaction is maintained at least about 7.5 prior to contacting theplasma sample with the solution of the preparing step.

In another embodiment of the present invention, a method is provided fordetermining Lp-PLA2 enzyme activity in a tissue sample obtained from ananimal comprising the steps of:

-   -   a) contacting 110 μL of a solution comprising:        -   a solution comprising 90 mM            1-myristoyl-2-(4-nitrophenylsuccinyl) phosphatidylcholine            contacted with a solution comprising 200 mM HEPES, 200 mM            NaCl, 5 mM EDTA, 10 mM CHAPS, 10 mM sodium 1-nonanesulfonate            at a pH 7.6 in a ratio of 0.66 μL to 110 μL; with at least            one 254 tissue sample from an animal; with 25 μL each of a            p-nitrophenol standard solution comprising; 4, 3, 2, 1, 0.4            or 0.2 nmol/μL p-nitrophenol in methanol; and 25 μL of            phosphate buffered saline (PBS) or ddH₂O to make a blank;            and    -   b) determining Lp-PLA2 activity.

In one aspect, the tissue sample from animal is blood plasma. In anotheraspect, the animal is human. In yet another aspect, the animal has beenadministered an inhibitor of Lp-PLA2 prior to obtaining the tissuesample. Inhibition of Lp-PLA2 enzyme activity by said Lp-PLA2 inhibitoradministered prior to obtaining said tissue sample is measured bycomparing Lp-PLA2 activity of a tissue sample free of said Lp-PLA2inhibitor.

In another embodiment of the present invention, a method is provided fordetermining Lp-PLA2 enzyme activity in a tissue sample obtained from ananimal wherein enzyme activity is determined by:

-   -   a) generating a standard curve by plotting optical density (OD)        values at 405 nm for the p-nitrophenol standard solutions vs.        p-nitrophenol (nmol/well);    -   b) calculating the slope (OD/nmol) of the standard curve;    -   c) calculating aborbance change between 3 and 1 minute        (ΔOD_(3min-1min)) for both solutions comprising tissue samples        and blank; and    -   d) calculating Lp-PLA2 activity using the following formula:        Lp-PLA2 activity(nmol/min/ml)=(ΔOD _(sample) −ΔOD        _(blank))÷slope(OD/nmol)÷0.025 ml÷2 minutes.

In another embodiment of the present invention, a method is provided fordetermining Lp-PLA2 enzyme activity in a tissue sample obtained from ananimal comprising the steps of:

-   -   a) preparing a solution comprising 200 mM HEPES, 200 mM NaCl, 5        mM EDTA, 10 mM CHAPS, 10 mM sodium 1-nonanesulfonate at a pH        7.6;    -   b) preparing a solution comprising 90 mM        1-myristoyl-2-(4-nitrophenylsuccinyl) phosphatidylcholine;    -   c) preparing 100, 75, 50, 25, 10 and 5 nmol/μL stock solutions        of p-nitrophenol in methanol;    -   d) preparing working solutions for p-nitrophenol standards by        diluting 40 μL of stock solutions of step c into 960 μL of        methanol;    -   e) contacting the solution of step b and the solution of step a        in a ratio of 0.66 μL to 110 μL to make an assay buffer;    -   f) adding 120 μL of assay buffer to each well in a 96-well        V-bottom plate;    -   g) adding 25 μL of each p-nitrophenol standard working solution        of step d into a separate well of two columns of a 96-well        flat-bottom plate;    -   h) adding 25 μL of tissue sample from an animal per well that do        not contain p-nitrophenol standards of the flat-bottom plate of        step g;    -   i) adding 25 μL of PBS or dd H₂O into an empty well in the        flat-bottom plate for use as a blank;    -   j) contacting 110 μL of assay buffer from the V-bottom plate to        each well of the flat-bottom assay plate;    -   k) placing the flat bottom assay plate onto a plate reader and        reading at 405 nm;    -   l) generating a standard curve by plotting optical density (OD)        values for the standard solutions vs. p-nitrophenol (nmol/well);    -   m) calculating the slope (OD/nmol) of the standard curve;    -   n) calculating absorbance change between 3 and 1 minutes        (ΔOD_(3min-1min)) for both test samples and the blank; and    -   o) calculating Lp-PLA2 activity using the following formula:        Lp-PLA2 activity(nmol/min/ml)=(ΔOD _(sample) −ΔOD        _(blank))÷slope(OD/nmol)÷0.025 ml÷2 minutes.

Calculating the absorbance change can be performed at various intervalsincluding, but not limited to, 2 and 0 minutes, 1 and 0 minutes andabout 15-second intervals measured over about a 10 minute reaction time.

The following examples illustrate various aspects of this invention.These examples do not limit the scope of this invention which is definedby the appended claims.

EXAMPLES

Unless otherwise indicated all plasma samples were collected from humanand are human plasma. Unless otherwise indicated, plasma samples for thefollowing examples were collected as follows. Blood was collected intoEDTA-containing tubes. The tubes were centrifuged at 1730×g for 10minutes. Plasma was drawn off with transfer pipettes into tubes andstored at −80° C.

In experiments in which Lp-PLA2 inhibitor was added to tissue samples invitro the following procedure was used, unless otherwise indicated. A 9mg/mL stock solution was prepared in PBS. A series of working dilutionswere prepared in PBS to give concentrations of 90000, 9000, 6000, 3000,1000, 500, 200, 100, and 0 ng/mL. One microliter of each workingdilution was added to every 100 ul of plasma or serum followed byincubation at 37° C. for 1 hour. The final concentrations of Lp-PLA2inhibitor in plasma or serum were: 900, 90, 60, 30, 10, 5, 2, 1, and 0ng/mL.

Example 1 The Auto PAF AH Assay Kit

The Auto PAF AH assay kit, manufactured by Azwell (Osaka, Japan), iscommercially available in the United States through Karlan ResearchProducts Corporation (Santa Rosa, Calif.). This assay was evaluated onan Olympus Au640 clinical chemistry analyzer and is described in thisExample 1.

Materials

Azwell Auto PAF-AH Assay Kit:

-   -   R1: 200 mM HEPES, 200 mM NaCl, 5 mM EDTA, 10 mM CHAPS, 10 mM        sodium 1-nonanesulfonate, pH 7.6    -   R2A: 20 mM citric acid monohydrate, 10 mM sodium        1-nonanesulfonate, pH 4.5    -   R2B: 90 mM 1-myristoyl-2-(4-nitrophenylsuccinyl)        phosphatidylcholine        Assay Procedure

-   1. Enter assay parameters from the following table into the Olympus    Au640 analyzer, and create a PAF AH assay program:    -   Sample volume: 2 μL    -   Reagent 1: 240 μL    -   Reagent 2: 80 μL    -   Wavelength (main): 410 nM    -   Wavelength (sub): 480 nm    -   Method: Rate    -   Point 1 (FST): 14    -   Point 1 (LST): 21    -   Calibration Type: MB    -   Formula: Y=AX+B    -   Counts: 2    -   MB Type Factor: 11595

-   2. Prepare the following reagents:    -   R1: Use this buffer solution as supplied in Azwell Auto PAF AH        assay kit. Store at 4° C. Protect from light.    -   R2: Prepare the R2 working solution by mixing R2A and R2B        (supplied in Azwell Auto PAF AH assay kit) in the proportion of        19:1. Store at 4° C. Protect from light.

-   3. Aliquot 30 μL or more of each plasma sample into a 2 mL Sarstedt    micro-tubes (Sarstedt Incorporation, part No. 72.694.007). Briefly    centrifuge to spin down fibrin clots/particles in the plasma.

-   4. Place Sarstedt tubes containing plasma samples onto sample tubes    that fit the instrument. Run plasma samples through the Au640    analyzer. After choosing the PAF AH assay program, the analytical    procedure described below is performed automatically:    -   Test sample (2 μL)+R1 (240 μL), 37° C., 5 minutes [0-5 minutes]    -   Add R2 (80 μL), 37° C., 5 minutes [5-10 minutes]    -   Measure the absorbance at 410 nm and 480 nm [6-8 minutes]    -   Calculate PAF AH activity (IU/L)

-   5. Include Bio-Rad Lyphochek Assayed Chemistry Control Level 1 and    Level 2 (C-310-5 and C-315-5, Bio-Rad, Hercules, Calif.) as quality    controls in each run. The Lp-PLA2 activity values for these two    controls are within the range of normal human plasma Lp-PLA2.

Example 2 High Throughput Radiometric Assay for Measurement of Lp-PLA2Activity

A high throughput radiometric assay was developed for measuring Lp-PLA2activity in a sample. This assay is fully described in WO2005/001416. Asummary of a high throughput radiometric activity assay is provided inthis Example 2.

Equipment

-   Scintillation Counter TopCount Microplate Scintillation and    Luminescence Counter, Perkin-Elmer (formerly Packard), CA-   Centrifuge Allegra 25R benchtop centrifuge, Beckman Coulter, Calif.-   Plate shaker Lab-Line Titer Plate Shaker (VWR cat #57019-600)-   Oven Barnstead/Thermolyne, series 9000, temperature range 10-250° C.    (VWR cat#52205-065)-   12-channel Pipettors BRAND Transferpette®-12, BrandTech Scientific,    Inc., Essex, Conn.    Material-   Polypropylene Plates Costar*Brand 96-Well Plates, Polypropylene,    Nonsterile, Without Lids, Costar 3365, Corning, Inc., Corning, N.Y.    (VWR cat #29444-104)-   PicoPlate Plates 96-Well white solvent-resistant microplates, Perkin    Elmer Life Sciences, Inc, Boston, Mass. (cat #6005162)    Reagents    -   HEPES (4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid)        Sigma Chemical Co., St. Louis Mo. (Cat # H9897100)    -   Sodium Chloride, Sigma Chemical Co., St. Louis Mo. (Cat # S5150;        5.0 M)    -   EDTA, Sigma Chemical Co., St. Louis Mo. (Cat # E7889; 0.5 M)    -   ³H-Platelet Activating Factor, 1-O-Hexadecy[acetyl-³H(N)],        (³H-PAF)-NEN Life Science Products, Roxbury, Mass. (Cat #        NET-910, supplied as an ethanol solution, typically 0.1 mCi/mL;        250uCi)    -   C16-PAF, (1-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine):        Avanti Polar Lipids, Alabaster, Ala. (Cat #878110; 5.0 mg/ml)    -   MicroScint-20: Perkin Elmer Biosciences, Boston, Mass. (cat        #6013621)    -   Fatty acid-free bovine serum albumin (BSA): Sigma Chemical Co.,        St. Louis Mo. (Cat # A0281; 1.0 gm)    -   Trichloroacetic acid (TCA): Sigma Chemical Co., St. Louis Mo.        (Cat # T9159)        Assay Buffer    -   100 mM Hepes, pH 7.4    -   150 mM NaCl    -   5 mM EDTA    -   Store it at room temperature        Procedures-   1. Prepare a ³H-PAF working solution (for 100 reactions):    -   a) Aliquot 480 μl ³H-PAF (10 μM=0.1 mCi/ml at 10.0 Ci/mmol) and        125.3 μl of [C16]PAF (5.0 mg/ml; MW: 524) to a tube;    -   b) Mix and air dry in the hood;    -   c) Resuspend the dried pellets in 12.0 ml of assay buffer giving        working solutions of 100 μM PAF (i.e. ³H-PAF at 0.4 μM and cold        [C16]PAF at 99.6 μM);-   2. Aliquot 5 μL of assay buffer (for Total counts and Blanks; n=8)    or plasma samples in duplicates into a 96-well plate;-   3. Equilibrate the plate to 21° C.;-   4. Add 100 μL of the ³H-PAF solution to each well, mix and incubate    the plate at 21° C. for 5 minutes;-   5. Add 50 μL of ice-cold BSA solution (50 mg/ml) to all wells, mix    and incubate the plate in a refrigerator for 5 minutes;-   6. Add 25 μL of ice-cold TCA solution (56%) to each well, mix and    incubate the plate in a refrigerator for 15 minutes;-   7. Centrifuge the plate at 6,000 g for 15 minutes at 4° C.;-   8. Aliquot 45 μL of the supernatants to a 96-well polystyrene plate;-   9. Add 10 μl, of ³H-PAF working solution to 6 Total Counts wells;-   10. Add 200 μL of MicroScint-20 scintillation cocktail to each well;-   11. Cover the plate with a plate tape and vortex mix at max speed    for 10 minutes;-   12. Get static off the plate by wiping with a wet tissue and drying    with another clean one;-   13. Count with a TopCount scintillation counter for 2 minutes each;    and-   14. Calculate Lp-PLA2 activity:    Lp-PLA2 activity(nmoles/min/ml)=160*(CPM _(45μl-supe) −CPM    _(Blanks))/(CPM _(10μl-spiking) −CPM _(Blanks))    -   -   Where CPM_(45μl-supe) is the average count from each sample            CPM_(Blanks) is the average count of the Blanks

    -   CPM_(10μl-spiking) is the average count of the Total Counts

Example 3 Correlation of Auto PAF AH assay and High ThroughputRadiometric Assay

A panel of 120 plasma samples from healthy human volunteers was assayedfor Lp-PLA2 activity at three clinics using the high-throughputradiometric assay described in Example 2. The same sample panel wasassayed using Azwell's Auto PAF AH assay described in Example 1 on theOlympus Au640 analyzer. Correlation was obtained against data generatedon the same panel of samples by the high throughput radiometric assay.Correlation coefficients (r) were 0.96, 0.94, and 0.95 for Auto PAF AHvs. the radiometric activity assay at the three clinics, respectively.The average CV between duplicates was 2.14% for the Auto PAF AH assay.

Example 4 Low Throughput Radiometric Assay

A low throughput radiometric assay capable of measuring Lp-PLA2 activityis provided below.

Materials

Scintillation Vials Wheaton Omni Vials, Millville, NJ (Cat # 225402)Scintillation Fluid EcoLite ™, ICN, Costa Mesa, CA (Cat # 882475)Equipment

Beta Counter Beckman Liquid Scintillation Counter, LS 5000TA, BeckmanInstruments, Fullerton, CA Water Bath Fisher Scientific, Edison, NJMicrocentrifuge Jouan Inc., Winchester, VA, Model No. A-14Reagents

HEPES (4-(2-hydroxyethyl)- Sigma Chemical Co., St. Louis MO1-piperazineethane sulfonic (Cat # H 9136) acid) Sodium Chloride SigmaChemical Co., St. Louis MO (Cat # S 7653) Chloroform Aldrich ChemicalCo., Milwaukee, WI (Cat # 36,692-7) Methanol Aldrich Chemical Co.,Milwaukee, WI (Cat # 27,047-4) ³H-Platelet Activating NEN Life ScienceProducts, Roxbury, MA Factor, 1-O-Hexadecyl-[acetyl- (Cat # NET-910,supplied as an ethanol ³H(N)], (³H-PAF) solution, typically 0.1 mCi/mL)C16-PAF, (1-hexadecyl-2- Avanti Polar Lipids, Alabaster, AL (Cat #acetyl-sn-glycero-3- 878110, supplied as 5 mg/mL CHCl₃ phosphocholine)solution)Assay BufferHEPES/NaCl Buffer:

50 mM HEPES, 150 mM NaCl, pH 7.4 at 37° C.

Assay Solutions

³H-PAF Working Solution:

Pipette 5 μCi (typically 50 μL of the solution supplied by the vendor)of ³H-PAF stock solution into a 1.4 mL glass vial. Add 340 ug (68 μL ofa 5 mg/mL solution) of C16-PAF. Evaporate to dryness under a gentlestream of nitrogen gas in a fume hood. Reconstitute with 1.3 mL ofHEPES/NaCl buffer. This will prepare sufficient working solution forapproximately 62 assay tubes.

Assay Master Mix:

In a 15 mL polypropylene tube, combine 7.3 mL of HEPES/NaCl buffer and1.1 mL of ³H PAF working solution. In the final reaction mixture, afteraddition of the plasma sample, the final concentration of PAF(unlabelled C16-PAF+³H-PAF) is 50 uM (the 200 μL reaction volumecontains 10 nmols PAF).

For testing the inhibition of LpPLA2 activity in plasma, the assay wasperformed as follows:

(1) 1104 HEPES/NaCl buffer+20 μL of appropriate working dilution ofLp-PLA2 inhibitor+50 μL of plasma sample were added to a 1.5 mLmicrocentrifuge tube and incubated at 37° C. for 15 minutes.

(2) 20 μL of 3H-PAF working solution was added and the samples wereincubated at 37° C. for 30 seconds.

(3) Reactions terminated by the addition of 600 μL of CHCl₃/CH₃OH andprocessed by the assay procedures described herein.

Assay Procedures

-   1. Thaw plasma samples and place in 37° C. water bath to temperature    equilibrate.-   2. Add 150 μL of assay master mix to 1.5 mL polypropylene tubes and    place in 37° C. water bath. Allow 5 minutes for temperature    equilibration.-   3. Add 50 μL of plasma sample or 50 μL of HEPES/NaCl buffer for    buffer blanks (all samples are assayed in duplicate) to appropriate    tubes containing assay master mix, vortex briefly, and incubate for    30 seconds in the 37° C. water bath.-   4. Stop reaction by addition of 600 μL of CHCl₃/CH₃OH solution and    vortex well.-   5. Just prior to centrifuging, briefly re-vortex the samples.    Separate organic and aqueous phases by centrifugation in a    microcentrifuge at maximum speed for 2 minutes.-   6. Collect 250 μL of the upper, aqueous phase and transfer to a new    1.5 mL polypropylene tube.-   7. Add 250 μL of CHCl₃ and vortex well.-   8. Separate organic and aqueous phases by centrifugation in a    microcentrifuge at maximum speed for 1 minute.-   9. Collect 150 μL of the upper, aqueous phase and transfer to a 7 mL    scintillation vial.-   10. Add 2 mL of EcoLite™ or equivalent liquid scintillation fluid.-   11. Count samples in liquid scintillation counter using a counting    program that has been set up to determine cpm, counting efficiency,    and dpm.-   12. For determination of total radioactivity in the reaction,    duplicate 150 μL aliquots of the assay master mix are counted.    Data Reduction and Analysis

Either cpm or dpm values may be used for calculation of Lp-PLA₂activity. If the counting efficiency is the same for the samples, bufferblanks, and total radioactivity vials, cpm values may be used. Ifdifferent counting efficiencies are observed, dpm values should be used.For all of the results in this report, dpm values were used for activitycalculations. The following equation is used to calculate LpPLA₂activity (reported as nmols/min/mL) from the raw data:((x−y)÷z)×40where,

-   x=cpm (or dpm) of plasma sample×1.65 (This corrects for the total    volume of the aqueous phase in each extraction. This correction is    necessary since only a portion of the aqueous phase is collected    after each of the extractions.)-   y=cpm (or dpm) of buffer blanks×1.65 (average of duplicate    determinations)-   z=cpm (or dpm) of total radioactivity samples divided by 10 (there    are 10 nmols of PAF in each reaction tube (average of duplicate    determinations)-   40=factor to adjust results to nmol/min/mL (each reaction is for 30    seconds and the volume of plasma used in each reaction is 50 uL)

Example 5 Comparison of Inhibition of Lp-PLA2 Activity Measured by theAuto PAF AH assay and Low Throughput Radiometric Assay

Plasma was collected from six human subjects at different timepointsafter in vivo drug administration of an Lp-PLA2 inhibitor during aclinical trial. Subjects #17 and #18 were dosed with 120 mg of FormulaI, described below, subjects #24 and #25 with 180 mg, and subjects #21and #22 with 240 mg. Subjects #21 and #25 also received placebo on adifferent day. Lp-PLA2 activity was measured by the low throughputradiometric assay, described in Example 4, and >90% inhibition wasobserved with all six drug-treated subjects. However, Lp-PLA2 inhibitionwas not apparent when measured by the Auto PAF AH assay, as described inExample 1. The Auto PAF AH assay is insensitive to in vivo druginhibition of Lp-PLA2. See Table 1 below.

TABLE 1 Measurement of Lp-PLA2 Activity in Patients Who ReceivedInhibitor in vivo Lp-PLA2 Activity (nmol/min/mL) LTP Radiometric AssayAuto PAF AH Assay Time Drug Drug Drug Drug Drug Drug Drug Drug Drug DrugDrug Drug (hr) #17 #18 #21 #22 #24 #25 #17 #18 #21 #22 #24 #25 0 36.1929.82 16.03 32.14 39.13 19.08 402 349 197 345 459 205 0.5 34.25 3.708.19 30.25 35.38 18.86 346 303 192 353 398 177 1 17.33 2.10 1.39 27.433.53 2.52 410 260 239 345 426 178 2 3.82 1.30 0.62 8.76 1.48 0.71 338296 214 332 336 178 3 2.04 1.77 0.79 4.61 1.11 0.51 333 299 217 357 482196 4 1.83 1.82 0.88 1.58 1.02 0.45 333 297 206 350 502 194 6 1.22 2.331.06 1.18 1.49 0.53 297 295 184 350 402 186 12 3.38 4.72 2.19 2.91 3.261.56 321 295 197 353 538 157 24 6.05 7.05 3.87 5.06 6.65 3.46 413 323235 362 547 229 32 7.31 6.55 3.17 3.59 7.85 4.15 346 296 242 350 530 21348 10.62 9.64 5.34 5.82 10.29 5.42 475 287 211 321 537 221 96 18.3114.65 10.22 12.38 16.88 11.05 463 322 227 341 569 245 144 29.31 18.5114.26 17.72 25.14 17.14 452 324 224 369 502 313

Inter-run and within-run variability for the Auto PAF AH assay on theOlympus Au640 has been consistently low with CV less than 5% betweenreplicates. In this experiment, the average CV between duplicates was 2%for placebo samples and 3% for all drug samples. However, Lp-PLA2activity measured by the Auto PAF AH assay fluctuated over time for bothdrug and placebo subjects. Similarly, radiometric activity values forthe placebo subjects fluctuated over time with a higher % CV comparedwith the Auto PAF AH assay. See Table 2 Observed variability in Lp-PLA2activity for the placebo subjects appears to be biological variability.

TABLE 2 Lp-PLA2 Activity (nmol/min/mL) in Patients who Received Placeboand Inhibitor in vivo Lp-PLA2 Activity (nmol/min/mL) LTP RadiometricAuto PAF AH Auto PAF AH Time (hr) Placebo #21 Placebo #25 Placebo #21Placebo #25 Drug #21 Drug #25 0 23.50 22.10 231 238 197 205 0.5 26.1523.11 227 246 192 177 1 18.97 23.69 237 246 239 178 2 25.99 27.10 233245 214 178 3 27.07 33.33 247 260 217 196 4 28.71 12.14 219 267 206 1946 25.31 24.97 216 232 184 186 12 25.54 25.11 238 252 197 157 24 28.4027.09 250 268 235 229 32 24.96 31.86 294 275 242 213 48 25.50 24.72 233279 211 221 96 14.34 23.38 256 347 227 245 144 27.03 30.30 271 247 224313 Mean 24.73 25.30 242.46 261.69 214.23 207.08 Stdv. 3.96 5.27 21.5729.37 18.62 40.01 % CV 16.00 20.85 8.89 11.22 8.69 19.32

Formula I, 2-(2-(3,4-Difluorophenyl)ethyl)-1H-quinoline-4-1-ylN-(4′-trifluoromethylbiphenyl-4-ylmethyl)-acetamide bitartrate; ispresented below and is described in WO 02/30904.

Example 6 Comparison of Inhibition of Lp-PLA2 Activity Measured by theAuto PAF AH assay and Low Throughput Radiometric Assay

Plasma samples were evaluated from eight subjects who received 100 mg ofa second Lp-PLA2 inhibitor during a clinical trial. The Lp-PLA2inhibitor used in the study,1-(N-(2-(Diethylamino)ethyl)-N-(4-(4-trifluoromethylphenyl)benzyl)-aminocarbonylmethyl)-2-(4-fluorobenzyl)thio-5,6-trimethylenepyrimidin-4-onebitartrate, is described below as Formula II and is described in WO01/60805:

Four of the eight subjects also received placebo on a different day.

Greater than 90% inhibition of Lp-PLA2 activity was observed using thelow throughput radiometric assay for in vivo administration of theLp-PLA2 inhibitor. However, no inhibition was measured with the Auto PAFAH assay (see Table 3). Lp-PLA2 activity values fluctuated for both thedrug and placebo subjects as measured by Auto PAF AH assay apparentlydue to biological fluctuation.

TABLE 3 Inhibition of LP-PLA2 Activity as Measured by Auto PAF AH andLow Throughput Radiometric Assay. Timepoint (hr) Drug #24 Drug #25 Drug#26 Drug #27 Drug #28 Drug #29 Drug #30 Drug #31 LTP Radiometric Assay 020.48 20.11 25.74 24.56 23.95 30.95 25.58 23.13 0.5 6.74 10.08 1.8516.77 4.43 22.16 10.97 8.05 1 1.03 2.14 1.89 5.21 3.25 4.28 7.63 3.93 20.88 0.77 1.62 2.45 1.97 2.13 6.10 1.10 3 0.82 1.20 1.83 1.74 2.25 2.072.43 0.92 4 1.48 1.21 1.85 1.28 2.43 2.20 2.00 1.13 6 1.45 1.22 1.671.74 3.25 2.66 2.74 1.20 12 2.98 3.06 4.20 3.99 5.34 7.20 5.43 3.37 245.59 5.99 7.18 7.06 8.30 15.24 8.94 5.23 32 8.24 5.44 20.40 8.95 7.9432.93 10.40 7.39 48 10.06 7.62 20.18 13.29 11.24 27.30 13.81 8.56 7214.77 11.81 13.25 13.41 13.69 29.10 18.31 12.21 96 16.18 14.58 14.7916.19 15.59 27.66 19.75 15.46 Auto PAF AH Assay 0 370 370 370 370 370370 370 370 0.5 352 352 352 352 352 352 352 352 1 613 613 613 613 613613 613 613 2 356 356 356 356 356 356 356 356 3 373 373 373 373 373 373373 373 4 360 360 360 360 360 360 360 360 6 323 323 323 323 323 323 323323 12 369 369 369 369 369 369 369 369 24 375 375 375 375 375 375 375375 32 416 416 416 416 416 416 416 416 48 365 365 365 365 365 365 365365 72 435 435 435 435 435 435 435 435 96 445 445 445 445 445 445 445445

Example 7 Substrate Specificity Testing

A manual colorimetric Lp-PLA2 activity assay was developed using thesubstrate1-myristoyl-2-(p-nitrophenylsuccinyl) phosphatidylcholinemanufactured by Azwell (Osaka, Japan). This assay is a correspondingmicrotiter-plate version of the Auto PAF AH Assay compatible with aspectrophotometric plate reader. This manual assay was used to evaluatethe physical properties of the substrate. Presented here are data onsubstrate specificity.

Materials

-   -   R1: 200 mM HEPES, 200 mM NaCl, 5 mM EDTA, 10 mM CHAPS, 10 mM        Sodium 1-nonanesulfonate, pH 7.6, Store at 4° C.    -   R2A: 20 mM citric acid monohydrate, 10 mM sodium        1-nonanesulfonate, pH 4.5,    -   R2B: 1-myristoyl-2-(4-nitrophenylsuccinyl) phosphatidylcholine,        90 mM    -   p-nitrophenol: Sigma-Aldrich Chemical Co., St. Louis, Mo. (Cat        #1048-25G)    -   Ethanol: Sigma-Aldrich Chemical Co., St. Louis, Mo. (Cat #7023)    -   Methanol: VWR International, West Chester, Pa. (Cat #        EM-MX0482-6)        Reagent Preparation    -   R2: Mix R2A and R2B in ratio of 10:1. Store at 4° C. for no        longer than two weeks before use.    -   p-Nitrophenol standards: Make 1M of 4-nitrophenol solution in        Methanol. Dilute 100 μL, 75 μL, 50 μL, 25 μL, 10 μL, 5 μL of the        1M solution in 1 mL of Methanol to make 100, 75, 50, 25, 10, and        5 nmol/μL stock solution respectively. Make working solution for        each standard by diluting 100 μL of stock solution into 900 μL        of methanol (1:10 dilution). Store both stock and working        solution at 4° C.        Assay Procedure

-   1. Set temperature of the plate reader (SPECTRAmax® PLUS³⁸⁴UV/VIS    Microplate Spectrophotometer, Molecular Devices, Sunnyvale, Calif.)    at 21° C.

-   2. Add 120 μL of R1 into each well in a 96-well flat-bottom assay    plate (Costar 3595, Corning, Inc., Corning, N.Y.) using a    multi-channel pipettor.

-   3. Add 10 μL of p-nitrophenol standard working solution into each of    the duplicate wells in Column 1 and 2. Use 7 standard points for    generating a standard curve: 0, 5, 10, 25, 50, 75, 100 nmol/well.    Leave well 1H and 2H for blank controls.

-   4. Add 5 μL of plasma individually into each well. Use duplicate for    each sample. Set up blank controls by adding 5 μL of ddH₂O instead    of plasma into well 1H and 2H. Mix the plate well by hand.

-   5. Incubate the plate at 37° C. for 5 minutes.

-   6. Cool the plate at 21° C. in the plate reader for 5 minutes.

-   7. Take the plate out from the plate reader. Add 40 μL of R2 into    each well using a multi-channel pipettor, changing tips after each    addition. Time the start of R2 addition.

-   8. Add 2 μl of ethanol into each well using a multi-channel    pipettor, changing tips after each addition. The purpose of this    step is to rid of all the air bubbles generated in wells. The    duration between first R2 addition and plate reading in Step #9 is 4    minutes.

-   9. Read the plate at 405 nm for 20 minutes with a 2-minute interval.    Include a 2-minute auto-mixing before reading the plate.    Activity Calculation

-   1. Generate a standard curve by plotting average OD values at 0 and    20 minutes (OD_(0min) and OD_(20min)) for the 7 standards vs.    p-nitrophenol (nmol/well). Calculate the slope of the standard    curve.

-   2. Calculate AOD values for each blank well between 2 and 4 minutes    (OD_(4min)-OD_(2min)) and average the two ΔOD values for the blanks.

-   3. For each sample well, calculate ΔOD values between 2 and 4    minutes and then Lp-PLA2 activity    (nmol/min/ml)=(ΔOD_(sample)−ΔOD_(blank))÷slope (OD/nmol)÷0.005 ml÷2    minutes.

-   4. Calculate an average activity value for duplicate sample wells.    Results

Substrate specificity against Lp-PLA2 was assayed by using two Lp-PLA2inhibitor compounds; Formula II, which is described in Example 6, andFormula III, which is presented below:

Formula III or1-(N-(2-(Diethylamino)ethyl)-N-(4-(4-trifluoromethylphenyl)benzypaminocarbonylmethyl)-2-(4-fluorobenzyl)thio-5-(1-methylpyrazol-4-ylmethyl)pyrimidin-4-oneis described in WO 00/66567.

Plasma samples from four healthy patients were incubated in vitro withincreasing amount of Formula III. For addition of the inhibitor solutionto the reaction mixtures, a 100 mM stock solution was prepared in DMSO.A series of 1:10 working dilutions were prepared in DMSO to giveconcentrations which ranged between 10 mM and 0.01 nM. One microliteraliquots of each working dilution were added in each reaction. The finalconcentrations of Lp-PLA2 inhibitor were (in nM) 60,000, 6,000, 600, 60,6, 0.6, 0.06, 0.006, 0.0006, 0.00006 and 0.

All four plasma demonstrated decreasing Lp-PLA2 activity as shown inTable 4. Inhibition achieved by Formula III in all four samples reachedover 90%, comparable to the natural Lp-PLA2 substrate PAF used in theradiometric activity assay. Formula II also showed over 90% inhibitionof the substrate hydrolysis when incubated in vitro in the same fourplasma samples.

TABLE 4 In Vitro Inhibition of Lp-PLA2 Activity by Formula III in FourPlasma Samples Activity (nmol/min/mL) % Inhibition Drug (nM) #3 #7 #8#10 #3 #7 #8 #10 600 22.65 10.50 9.67 10.91 88.50 92.75 90.37 93.78 6016.25 12.64 0.14 14.17 91.75 91.28 99.86 91.92 6 48.61 31.39 21.81 35.9775.32 78.33 78.29 79.49 0.6 102.78 78.61 50.69 83.89 47.81 45.73 49.5252.18 0.06 167.36 119.17 83.33 148.89 15.02 17.74 17.02 15.12 0.006162.22 138.06 95.42 178.61 17.63 4.70 4.98 −1.82 0.0006 200.97 142.3686.67 179.17 −2.05 1.73 13.70 −2.14 0 196.94 144.86 100.42 175.42 0 0 00

The assay buffer used in above experiments has high content of detergent(7.5 mM CHAPS and 10 mM Sodium 1-nonanesulfonate). When detergent waseliminated from the assay, Formula III only inhibited about 65% ofhydrolysis activity in plasma sample #10. When detergent was added inthe parallel experiment inhibition of more than 95% was reached.Therefore, it appears that this substrate is specific to Lp-PLA2 onlywhen it is assayed in the presence of buffer comprising detergent, asshown in Table 5.

TABLE 5 Effect of Detergent on Substrate Specificity % Inhibition Drug(nM) with detergent without detergent 60000 96.01 68.01 6000 95.68 62.73600 95.06 61.30 60 87.94 55.03 6 76.92 54.97 0.6 69.90 46.52 0.06 48.5826.96 0.006 24.22 26.65 0.0006 19.18 19.88 0.00006 10.68 7.20 0 0 0

Example 8 Modified Drug Sensitive Colorimetric Assay for Measurement ofLp-PLA2 Activity

For the Auto PAF AH assay, plasma samples are diluted about 160-fold andthe substrate is used at a concentration higher than its Km. It appearsthat when the concentration of substrate is higher than its Km thesubstrate competes with drug bound to Lp-PLA2 and promotes drugdissociation from the enzyme. For instance, the substrate concentrationused in the Auto PAF AH assay is 1100 μM, which is more than 5 timeshigher than its Km (Km is about 200 μM when plasma is used as the enzymesource and assayed by Auto PAF AH protocol, see Example 1).Pre-incubation of plasma with buffer R1 in Auto PAF AH assay alsoappears to promote drug dissociation before the start of assay reaction.Therefore, the assay of the present invention was modified by usinghigher plasma sample volumes and lower substrate concentrations comparedwith the Auto PAF AH assay. Additionally, the pre-incubation step ofplasma with R1 prior to substrate addition was eliminated. Moreover,elimination of buffer R2A increased reaction rates, which in turnenabled the use of lower substrate concentrations and a shorter assayincubation time during which drug dissociates compared with the Auto PAFAH assay

Materials

-   -   R1: 200 mM HEPES, 200 mM NaCl, 5 mM EDTA, 10 mM CHAPS, 10 mM        sodium 1-nonanesulfonate, pH 7.6    -   R2B: 90 mM 1-myristoyl-2-(4-nitrophenylsuccinyl)        phosphatidylcholine p-nitrophenol: Sigma-Aldrich Chemical Co.,        St. Louis, Mo. (Cat #1048-25G)        Reagent Preparation

-   Assay buffer: Mix R2B and R1 in a ratio of 0.66 μL to 110 μL. Store    on ice or at 4° C. Prepare immediately before use.

-   p-Nitrophenol standards: Prepare 1 M p-nitrophenol in methanol.    Dilute 100, 75, 50, 25, 10 and 5 μL of 1 M p-nitrophenol to 1 mL in    methanol to prepare 100, 75, 50, 25, 10 and 5 nmol/μL stock    solutions, respectively. Prepare working solutions for each standard    by diluting 40 μL of stock solution into 960 μL of methanol (1:25    dilution). Store stock and working solutions at 4° C.    Assay Procedure

-   1. Add 120 μL of assay buffer to each well in a 96-well V-bottom    plate (Costar 3897, Corning, Inc., Corning, N.Y.) using a    multi-channel pipettor or robot.

-   2. Add 25 μL of p-nitrophenol standard working solution into    duplicate wells in columns 1 and 2 on another 96-well flat-bottom    plate (Costar 9017, Corning, Inc., Corning, N.Y.). Use 7 standard    points for generating a standard curve: 0, 5, 10, 25, 50, 75, 100    nmol/well. Add 25 μL of PBS into well 1H and 2H for blank controls.

-   3. Briefly centrifuge plasma to spin down fibrin clot/particles. Add    25 μL of plasma per well in columns 3-12 on the same flat-bottom    plate containing p-nitrophenol standards. Use duplicates for each    sample.

-   4. Use a multi-channel pipettor or a robot to transfer 110 μL of    assay buffer from the V-bottom plate to the flat-bottom assay plate    containing plasma samples and p-nitrophenol standards. A Zymark    RapidPlate (Caliper Life Sciences, Hopkinton, Mass.) can perform    this step without generating bubbles in the wells. Other transfer    methods may generate bubbles due to the high detergent content of    R1. A small volume of ethanol can be used to eliminate air bubbles.

-   5. Immediately place the assay plate onto the plate reader    (SPECTRAmax® PLUS³⁸⁴ UV/VIS Microplate Spectrophotometer, Molecular    Devices, Sunnyvale Calif.) and auto-mix for 15 seconds.

-   6. Read the plate at 405 nm for 10 minutes at 15-second intervals at    room temperature. The duration between the start of enzymatic    reaction (addition of assay buffer to the assay plate) and    completion of the first absorbance reading is 1 minute.

The assay may be performed at room temperature. More stringenttemperature control may be required if room temperature fluctuateswithin or between labs.

Activity Calculation

-   1. Generate a standard curve by plotting average OD values at 0 and    10 minutes (OD_(0min) and OD_(10min)) for the seven standards vs.    p-nitrophenol (nmol/well). Calculate the slope of the standard    curve.-   2. Calculate Change in (ΔOD) values for each blank well between 1    and 3 minutes (OD_(3min)-OD_(1min)) and average the two ΔOD values    for the blanks.-   3. For each sample well, calculate ΔOD values between 1 and 3    minutes and then Lp-PLA2 activity    (nmol/min/ml)=(ΔOD_(sample)-ΔOD_(blank))÷slope (OD/nmol) 0.025 ml÷2    minutes.-   4. Calculate an average activity value for duplicate sample wells.

Example 9 Comparison of Radiometric Measurement Versus Modified DrugSensitive Colorimetric Measurement of Lp-PLA2 activity in the Presenceof Lp-PLA2 Inhibitor

Lp-PLA2 activity from blood plasma samples obtained from a healthy humansubject administered an Lp-PLA2 inhibitor was measured using the highthroughput radiometric assay described in Example 2 and the methods ofExample 8 with the following minor changes. The volume of plasma usedper well was 25 Substrate concentration was 1125 μM, and, 2 of substratesolution R2B was mixed in 40 μL of R2A before further mixing with 95 μLof R1 to make the assay buffer. Blood plasma samples were collected atfive timepoints after dosing (0.5, 1.0, 6.0, 48 and 96 hours postdosing). Both radiometric and colorimetric assays were used to determineLp-PLA2 activity as well as percent inhibition in each sample as shownin Table 6. As shown in Table 6, percent inhibition of Lp-PLA2 activityas measured by a radiometric assay showed peak inhibition as about 94%one hour after dosing while a modified drug sensitive colorimetric assayshowed peak inhibition at the 6-hour timepoint with about 64% inhibitionin activity. These data demonstrate that both methods can be used tomeasure the inhibition of Lp-PLA2 activity in samples obtained from ananimal that has been administered an Lp-PLA2 inhibitor. Blood samplesfrom humans are considered to be essentially free of Lp-PLA2 inhibitor96 hours post dosing.

TABLE 6 Comparison of Lp-PLA2 Activity as Measured Using Radiometricversus Modified Drug Sensitive Colorimetric Assay Time Radiometric AssayColorimetric Assay Point Activity % Inhibition Activity % Inhibition(hour) (nmol/min/mL) (96 hr-100%) (milliOD/min) (96 hr-100%) 0.5 28.0247.00 34.47 36.55 1 3.19 93.97 22.98 57.70 6 10.14 80.83 19.8 63.56 4844.52 15.80 33.76 37.86 96 52.87 0 54.33 0

Example 10 Testing of Plasma Samples from a Clinical Study for Lp-PLA2Inhibition

Four human subjects recruited in a clinical trial of a novel Lp-PLA2inhibitor, Formula I (see Example 5) received different doses of thedrug. Drug dose for Subject #13, #36, #24, and #41 was 80 mg, 120 mg,180 mg, and 240 mg, respectively. Plasma was collected at 0, 0.5, 1, and3 hours after drug administration. Lp-PLA2 activity of these plasmasamples was assayed by the low throughput radiometric assay described inExample 4, the Auto PAF AH assay, described in Example 1, and modifieddrug-sensitive colorimetric assay, which is described in this Example 8.While the radiometric activity assay indicated >90% inhibition ofLp-PLA2 activity 3 hours after dosing in all four subjects, the Auto PAFAH assay failed to indicate drug inhibition. However, a modifieddrug-sensitive colorimetric assay of Example 8 indicated 85-90% druginhibition as shown in Table 7.

TABLE 7 Percent Inhibition of Lp-PLA2 Activity in Plasma Samples fromSubjects Administered Lp-PLA2 Inhibitor % Inhibition Drug-SensitiveRadiometric Assay Auto PAF AH Assay Colorimetric Assay Time Pt. No. (hr)13 36 24 41 13 36 24 41 13 36 24 41 0 0 0 0 0 0 0 0 0 0 0 0 0 0.5 46.1245.06 9.58 26.68 5.60 8.38 6.26 8.60 32.82 45.62 6.73 18.41 1 81.7991.46 90.98 88.27 9.98 14.03 11.53 14.55 73.08 84.97 86.26 77.78 3 93.7494.36 97.32 96.02 7.11 10.93 10.36 15.70 85.64 85.3 89.15 88.37

Similarly, both the radiometric assay and modified drug-sensitivecolorimetric assay showed a measured time-dependent effect on Lp-PLA2activity after dosing with Lp-PLA2 inhibitor as shown in Table 8. Littleeffect on Lp-PLA2 activity was observed using the Auto PAF AH assay asshown in Table 8.

TABLE 8 Lp-PLA2 Activity in Plasma Samples from Subjects AdministeredLp-PLA2 Inhibitor Lp-PLA2 Activity (nmol/min/mL) Drug-SensitiveRadiometric Assay Auto PAF AH Assay Colorimetric Assay Time Pt. No. (hr)13 36 24 41 13 36 24 41 13 36 24 41 0 27.3 26.94 39.13 46.97 330.50605.00 511.50 274.50 96.9 83.84 177.63 176.2 0.5 14.71 14.8 35.38 34.44312.00 553.00 479.50 251.50 65.1 45.59 165.67 143.76 1 4.97 2.3 3.535.51 297.50 517.00 452.50 236.00 26.08 12.61 24.41 39.14 3 1.71 1.521.05 1.87 307.00 510.00 458.50 244.50 13.92 12.33 19.27 20.49

Although the activity values generated from radiometric and modifieddrug-sensitive colorimetric assay are different, correlation between thetwo assays is r=0.975 for these 16 clinical plasma samples. Therefore,modified drug-sensitive colorimetric assays, described herein, althoughusing the same substrate as the Auto PAF AH assay, demonstrated abilityto detect in vivo drug inhibition of Lp-PLA2 in drug-treated humansubjects, while the Auto PAF AH assay did not.

Example 11 Testing of Additional Plasma Samples from a Clinical Studyfor Lp-PLA2 Inhibition

Plasma samples were collected from ten subjects in a clinical trial forthe Lp-PLA2 inhibitor of Formula I. Subjects #109, #114, #115, #142 and#145 received 50 mg of Formula I while subjects #118, #119, #121, #123and #124 received 120 mg of the compound. Lp-PLA2 activity of theseplasma samples were assayed by the high throughput radiometric assay asdescribed in Example 4, the Auto PAF AH assay as described in Example 1,and modified drug-sensitive colorimetric assay as described in Example8. Consistently, the Auto PAF AH assay failed to measure drug inhibitionof Lp-PLA2 activity in these samples with maximal inhibition of 29%detected in subject #123. However, a modified drug-sensitivecolorimetric assay of Example 8 indicated comparable drug inhibitionwith radiometric assay in all subjects. Inhibition values for theradiometric assay and a modified, drug-sensitive colorimetric assayagreed within 15% for all but four time (#114/12 hr, #115/12 hr,#142/0.5 hr and #142/12 hr) as shown in Table 9.

TABLE 9 Lp-PLA2 Activity and Percent Inhibition by Subject and AssayLp-PLA2 Activity (nmol/min/mL) % Inhibition Drug- Drug- Time Auto PAFSensitive Auto PAF Sensitive Subject (hr) Radiometric AH ColorimetricRadiometric AH Colorimetric # 109 0 140.47 703.50 193.21 0.00 0.00 0.000.5 104.52 647.00 141.14 25.59 8.03 26.95 1 26.80 605.00 39.10 80.9214.00 79.76 2 14.51 559.50 30.57 89.67 20.47 84.18 3 18.06 602.50 35.0387.14 14.36 81.87 4 20.76 629.50 33.46 85.22 10.52 82.68 5 19.57 651.5031.77 86.07 7.39 83.55 6 21.78 642.00 66.08 84.49 8.74 65.80 9 32.01571.50 49.86 77.21 18.76 74.19 12 35.85 571.50 76.84 74.48 18.76 60.23#114 0 84.01 512.00 127.01 0.00 0.00 0.00 0.5 65.24 456.50 89.99 22.3410.84 29.15 1 18.68 414.50 24.96 77.76 19.04 80.35 2 14.34 406.50 21.2282.93 20.61 83.29 3 12.24 435.00 18.66 85.43 15.04 85.31 4 13.50 409.0018.15 83.93 20.12 85.71 5 14.14 385.00 16.58 83.17 24.80 86.94 6 16.10388.50 21.01 80.84 24.12 83.46 9 24.73 445.50 37.35 70.56 12.99 70.59 1228.82 409.50 111.00 65.69 20.02 12.60 #115 0 60.58 310.00 83.99 0.000.00 0.00 0.5 50.18 308.00 69.79 17.17 0.65 16.91 1 10.60 242.00 22.4682.50 21.94 73.26 2 10.00 282.00 16.31 83.49 9.03 80.58 3 13.03 287.5021.31 78.49 7.26 74.62 4 14.37 309.00 21.77 76.28 0.32 74.08 5 13.63279.50 15.83 77.50 9.84 81.16 6 15.96 306.50 25.84 73.65 1.13 69.24 925.91 289.50 36.27 57.23 6.61 56.82 12 26.92 336.00 53.72 55.56 −8.3936.04 #118 0 101.65 382.50 102.59 0.00 0.00 0.00 0.5 35.83 323.50 31.0864.75 15.42 69.70 1 16.73 334.00 19.69 83.54 12.68 80.81 2 13.70 313.5019.69 86.52 18.04 80.81 3 14.09 335.00 13.17 86.14 12.42 87.16 4 14.00346.50 7.30 86.23 9.41 92.88 5 14.05 353.50 22.94 86.18 7.58 77.64 615.68 330.00 20.65 84.57 13.73 79.87 9 21.11 338.00 27.16 79.23 11.6373.52 12 24.22 355.50 38.74 76.17 7.06 62.24 #119 0 141.40 736.00 180.590.00 0.00 0.00 0.5 25.97 596.50 20.14 81.63 18.95 88.89 1 16.04 568.0023.29 88.66 22.83 87.10 2 13.50 579.00 19.54 90.45 21.33 89.18 3 12.80616.50 20.61 90.95 16.24 88.59 4 12.85 597.50 21.27 90.91 18.82 88.22 511.69 748.00 21.36 91.73 −1.63 88.17 6 11.41 714.00 21.39 91.93 2.9988.16 9 18.51 578.50 27.87 86.91 21.40 84.57 12 21.27 607.50 38.46 84.9617.46 78.70 #121 0 97.09 440.50 134.39 0.00 0.00 0.00 0.5 16.07 402.0019.87 83.45 8.74 85.22 1 10.00 401.00 14.99 89.70 8.97 88.85 2 10.00402.50 12.22 89.70 8.63 90.90 3 10.00 414.50 15.08 89.70 5.90 88.78 410.00 389.50 16.18 89.70 11.58 87.96 5 10.00 416.00 18.14 89.70 5.5686.50 6 10.26 412.00 18.74 89.43 6.47 86.06 9 14.41 428.00 26.18 85.162.84 80.52 12 16.97 456.00 35.87 82.52 −3.52 73.31 #123 0 72.11 454.00116.90 0.00 0.00 0.00 0.5 39.68 410.50 63.95 44.97 9.58 45.29 1 19.83402.50 30.22 72.50 11.34 74.15 2 19.42 369.50 29.51 73.07 18.61 74.76 321.08 387.50 30.49 70.77 14.65 73.92 4 19.51 406.50 29.42 72.94 10.4674.84 5 20.24 428.50 35.46 71.93 5.62 69.67 6 19.66 393.00 39.29 72.7413.44 66.39 9 32.48 343.00 50.81 54.96 24.45 56.54 12 34.26 324.00 61.1652.49 28.63 47.69 #124 0 87.96 465.50 109.88 0.00 0.00 0.00 0.5 58.09434.50 64.40 33.96 6.66 41.39 1 12.39 429.50 18.74 85.91 7.73 82.95 210.00 367.50 10.11 88.63 21.05 90.80 3 10.00 362.50 11.51 88.63 22.1389.53 4 10.00 403.00 11.21 88.63 13.43 89.80 5 10.00 377.00 12.22 88.6319.01 88.88 6 10.00 366.00 17.01 88.63 21.37 84.52 9 11.21 387.00 21.0387.26 16.86 80.86 12 15.69 355.00 30.70 82.16 23.74 72.06 0 77.38 368.50100.48 0.00 0.00 0.00 #142 0.5 78.40 393.00 81.70 −1.32 −6.65 18.69 130.87 340.50 37.07 60.11 7.60 63.11 2 14.73 348.00 27.05 80.96 5.5673.08 3 12.41 348.00 22.64 83.96 5.56 77.47 4 11.47 338.50 19.97 85.188.14 80.12 5 10.66 336.50 18.35 86.22 8.68 81.74 6 12.93 325.00 25.1283.29 11.80 75.00 9 22.03 312.00 38.84 71.53 15.33 61.34 12 21.96 307.0044.60 71.62 16.69 55.61 #145 0 63.55 305.00 88.69 0.00 0.00 0.00 0.522.46 272.50 32.96 64.66 10.66 62.84 1 15.85 271.00 17.15 75.06 11.1580.66 2 14.45 271.50 21.07 77.26 10.98 76.24 3 11.99 277.50 20.89 81.139.02 76.45 4 10.02 256.50 19.02 84.23 15.90 78.55 5 10.14 273.50 20.7484.04 10.33 76.62 6 11.33 273.50 23.71 82.17 10.33 73.27 9 17.58 286.0033.36 72.34 6.23 62.39 12 20.83 261.50 34.83 67.22 14.26 60.73

Correlation of r=0.95 was obtained between a modified, drug-sensitivecolorimetric assay and the radiometric assay for the 100 samplesanalyzed in this study. The Auto PAF AH assay showed poor correlationwith radiometric assay in these drug dosed samples (r=0.31).

Example 12 Assay Dynamic Range

Instrument Low Limit of Quantitation

Twenty-five microliters of PBS were added into 110 μL of R1 containing0.67 μL of R2B. Sixteen replicates were prepared and randomly placed inwells across a microtiter plate. Absorbance at 405 mu was obtained andstandard deviation calculated between replicates. Six times standarddeviation (6×SD) was defined as the lower limit of quantitation for themicrotiter plate reader (SPECTRAmax® PLUS³⁸⁴ UV/VIS MicroplateSpectrophotometer, Molecular Devices, Sunnyvale, Calif.). The average ODreading from 16 replicates was 0.0437 with a standard deviation of0.0009. The lower limit of quantitation for the microtiter plate readerwas defined as 6×0.0009 or a change of 0.0054 OD units during assayincubation.

Linear Detection Range of p-Nitrophenol

Serial dilutions of p-nitrophenol were prepared in methanol. Twenty-fivemicroliters of p-nitrophenol at each concentration were added to 110 μlR1 (without R2B) in a microtiter plate. Absorbance values at 405 nm werelinear between 0.05 to 125 nmol of p-nitrophenol (r=0.996). However, theblank corrected absorbance of the 0.05 nmol p-nitrophenol sample wasonly 0.00415, which is below the microtiter plate reader's lower limitof quantitation of 0.0054 OD as defined above. Therefore, the lineardetection range of p-nitrophenol is set between 0.1 and 125 nmol ofp-nitrophenol per well.

Assay Dynamic Range

Assay dynamic range was defined by using both recombinant human Lp-PLA2protein (hrLp-PLA2) generated in-house and Lp-PLA2 protein purified fromhuman plasma in-house.

hrLp-PLA2 was serially diluted and 25 μL of each diluted hrLp-PLA2 wereassayed by a modified drug-sensitive colorimetric assay (data shown inTable 10). The second least amount of hrLp-PLA2 assayed 206 ng/mL showedan activity of 1.5 nmol/min/mL. Such level of activity would onlygenerate 0.075 nmol of p-nitrophenol in two minutes of substratehydrolysis reaction in the current assay configuration. Therefore, it islower than the linear detection range of the end product p-nitrophenol.hrLp-PLA2 greater than 13200 ng/mL showed plateau activity. The activityof hrLp-PLA2 between 412 to 13200 ng/mL demonstrated linearity with an Rvalue of 0.997. Therefore, the dynamic range of this assay appears to bebetween 4.4 and 397 nmol/min/mL, although lower and upper limits couldbe further defined. (see Table 10).

TABLE 10 Activity of Recombinant Human Lp-PLA2 Protein by ModifiedColorimetric Assay hrLp-PLA2 (ng/ml) Activity (nmol/min/mL) 0 0.0 1030.6 206 1.5 412 4.4 825 12.0 1650 30.0 3300 69.7 6600 169.0 13200 397.114666 413.7

hLp-PLA2 purified from plasma was also serially diluted and 25 pt ofeach dilution were assayed by a modified drug-sensitive colorimetricassay (data shown in Table 11). The activity of purified hLp-PLA2protein ranged between 6.25 to 1200 ng/mL demonstrated linearity with anR value of 0.97. Therefore, the dynamic range assessed using purifiedhLp-PLA2 appears to be between 2.47 and 363.60 nmol/min/mL, comparableto the one defined by hrLp-PLA2 (see Table 11). The relatively lowerupper limit of the dynamic range determined by purified hLp-PLA2 may beresulted from interference factors possibly present in the purifiedproduct and/or introduced during purification process. Limitedavailability of such purified protein prevents further investigation.

TABLE 11 Activity of Purified Human Lp-PLA2 Enzyme by Modified DrugSensitive Colorimetric Assay Purified hLp-PLA2 (ng/ml) Activity(nmol/min/mL) 0.00 0.00 1.56 0.65 3.13 1.37 6.25 2.47 12.50 5.06 25.0011.37 50.00 25.68 75.00 37.83 100.00 54.34 200.00 119.34 400.00 212.95600.00 273.60 800.00 324.85 1000.00 338.51 1200.00 363.60 1600.00 335.42

Example 13 Substrate Stability

Stability of the substrate in modified assay buffer (110 μL R1+0.67 μLR2B+25 μL PBS) was examined by monitoring absorbance changes every 15minutes over 120 minutes at room temperature. Although absorbanceincreased slowly but consistently over 2 hours reflecting gradualsubstrate degradation, the change in absorbance was only 0.002 OD unitsper 15 minutes. Therefore, substrate degradation appears to be moderateover 2 hours under a modified assay conditions. Since the assay takesonly 10 minutes to complete and activity is calculated based on a2-minute reaction period, absorbance changes from substrate degradationare insignificant and can be blank-corrected.

Example 14 Effect of Pre-Incubation of Human Plasma with Buffer R1 onDrug-sensitivity

In the Auto PAF AH assay, plasma is pre-incubated in buffer R1 at 37° C.for 5 minutes. This pre-incubation step may accelerate the dissociationof drug bound to Lp-PLA2 before the start of the reaction. To testwhether accelerated dissociation occurs, a plasma sample from a humansubject (#10) was incubated with increasing amount of Lp-PLA2 inhibitorat 37° C. for an hour. Twenty-five microliters of the in vitro FormularII drug-treated plasma was then pre-incubated with 100 μL R1 at roomtemperature for different times before running the assay for 10 minutesat room temperature after addition of 40 μL of R2 (final substrateconcentration of 1100 μM). Pre-incubation of plasma with R1 decreaseddrug-inhibition especially at lower drug concentrations. The highestlevel of drug inhibition was obtained when R1 and R2 were premixed andadded directly to plasma without pre-incubation, as shown in Table 12.Pre-incubation of plasma in R1 at 37° C. instead of room temperaturefurther deteriorates drug inhibition.

TABLE 12 Effect of Preincubation of Plasma in Buffer R1 on PercentInhibition of Lp-PLA2 Activity Preincubation of Plasma in R1 BufferReaction Time (minutes) Drug (ng/mL) 5 minutes 2 minutes 0 minutes R1R2premix 0 0.00 0.00 0.00 0.00 2 −2.33 3.94 10.76 15.10 5 2.39 4.83 17.1825.76 10 9.35 10.03 18.97 27.17 30 35.14 42.02 45.19 53.92 60 39.9739.17 49.00 57.64 90 72.26 72.58 76.61 77.69

Example 15 Effect of Substrate Concentration on Drug-Sensitivity

The substrate concentration is 1100 μM in the Auto PAF AH assay, whichis more than 5 times higher than its Km (Km=200 μM when plasma is usedas the enzyme source and assayed by Auto PAF AH protocol). Highsubstrate concentrations may compete with drug binding to Lp-PLA2. Totest this possibility, 25 μL of in vitro Lp-PLA2 inhibitor Formular IItreated human plasma samples were added to premixed R1 (100 μL) and R2(40 μL) containing different amounts of the substrate. Substratehydrolysis was immediately monitored at room temperature for 10 minutes.Lower substrate concentrations indicate greater drug inhibition.Activity values approached the lower limit of quantitation at the higherdrug levels when the substrate was used at 154 μM or less due to slowerhydrolysis rates. Consequently, the substrate concentration should bemaintained slightly above its Km in order to drive rapid substratehydrolysis while maintaining drug inhibition levels, as shown in Table13.

TABLE 13 Effect of Substrate Concentration on Percent Inhibition ofLp-PLA2 Activity Substrate Concentration (uM) Drug (ng/mL) 275 550 11002200 0 0.00 0.00 0.00 0.00 2 32.43 26.84 18.32 17.33 5 31.02 34.92 16.5330.96 10 51.38 50.23 25.26 42.68 30 67.57 56.99 46.00 28.30 60 76.8872.98 59.86 52.77 90 86.12 81.83 71.89 70.69

Example 16 Effect of Human Plasma Sample Volume on Drug-Sensitivity

Two μL of plasma were assayed in a 320 μL reaction for the Auto PAF AHassay, which corresponds to a plasma dilution factor of 160-fold. Highplasma dilution may promote drug dissociation from Lp-PLA2.Consequently, 5 to 50 of an in vitro Lp-PLA2 inhibitor Formula IItreated plasma sample were diluted with varying volumes of R1 and 40 μLof R2 to a final volume of 165 μL containing 1100 μM substrate.Hydrolysis was immediately monitored at room temperature for 10 minutes.Greater drug inhibition was observed with higher plasma sample volumes,as shown in Table 14.

TABLE 14 Effect of Sample Volume on Percent Inhibition of Lp-PLA2activity Plasma Sample Volume (μL) Drug (ng/mL) 5 15 25 50 0 0.00 0.000.00 0.00 2 88.47 4.15 18.32 13.22 5 09.11 19.85 16.53 10.26 10 −14.7339.11 25.26 33.03 30 33.90 23.53 46.00 55.58 60 34.14 56.68 59.86 67.6890 43.78 62.68 71.89 79.14

Example 17 Effect of Deletion of Buffer 2A on Drug-Sensitivity

In the Auto PAF AH assay, the substrate stock solution R2B is premixedin buffer R2A (20 mM citric acid monohydrate, 10 mM sodium1-nonanesulfonate, pH 4.5), which acts as a substrate stabilizer. Thesubstrate, after diluted in R2A, remains stable at 4° C. for 14 days.Faster hydrolysis rate was observed when R2A was omitted from the assay.For example, a colorimetric assay was performed with 180 nmol ofsubstrate (2 μL of R2B) and either 25 μL or 50 μL of plasma.Additionally, samples contained either 0 μL or 40 μL of R2A. Allreactions were diluted to either 125 μl or 165 μL with R1. Buffercomponents were pre-mixed and the reaction was initiated upon humanplasma addition. Substrate hydrolysis was immediately monitored at roomtemperature for 10 minutes. Vmax (milliOD/min) was calculated andcompared among different conditions. Higher hydrolysis rates wereobserved upon omission of R2A, independent of plasma volume as shown inTable 15.

TABLE 15 Effect of the Deletion of Buffer R2A from the Assay Sample 1 23 4 5 6 R1 100 μL  140 μL  100 μL  75 μL 115 μL  75 μL R2A 40 μL 0 μL 0μL 140 μL  0 μL  0 μL R2B  2 μL 2 μL 2 μL  2 μL 2 μL  2 μL Plasma 25 μL25 μL  25 μL  50 μL 50 μL  50 μL Vmax 40 90 70 40 150 120 (milliOD/Minute)

Since R2A has low pH of 4.5 compared with the other assay buffercomponents, whether addition of R2A affected the pH of the assayreaction was determined. The pH of assay reactions containing 110 μL ofR1, 0.66 μL of R2B and 25 μL of either plasma or ddH₂O were 7.52 and7.53, respectively. The pH dropped to 7.43 and 7.42, respectively, when40 μL of R2A were added to these assay samples. The effect of R2A onLp-PLA2 hydrolysis rates of the substrate is probably more thanpH-related.

Elimination of R2A from the assay increased hydrolysis rates, therebyallowing the use of lower substrate concentrations and shorter assayincubation times, both of which lower drug dissociation. Lp-PLA2activity values approached the lower limit of quantitation when 25 μL ofplasma were measured using 154 μM of substrate and R2A as described inExample 14. A substrate titration experiment was repeated using 50 μL ofin vitro Formula II-treated plasma (subject #10) and 75 μL of assaybuffer containing only R1 and R2B (no R2A). Assays were monitored atroom temperature for 10 minutes at 405 nm and Vmax and drug inhibitionwere calculated. Hydrolysis activity exceeded the lower limit ofquantitation at 900 and 9000 ng/mL of drug even at substrateconcentrations as low as 65 μM (see Table 16). Consequently, R2A waseliminated and lower substrate concentrations were incorporated in amodified colorimetric activity assay.

TABLE 16 Effect of Substrate Concentration on Vmax of SubstrateHydrolysis Vmax (milliOD/min) of Substrate Hydrolysis in Absence ofBuffer R2A Drug Substrate Concentration (uM) (ng/mL) 273 205 154 115 8665 9000 10.18 13.01 5.70 5.76 5.23 3.40 900 14.25 10.45 6.66 5.32 4.544.06 90 98.56 72.58 64.27 61.08 53.56 44.88 30 117.89 94.33 83.66 75.2165.47 54.08 10 114.89 93.75 79.87 76.21 67.31 54.90 5 112.58 93.22 82.1176.71 66.46 52.99 0 110.82 90.18 80.40 73.58 63.73 56.30

Earlier studies indicated higher drug inhibition as the substrateconcentration was lowered over 2200 μM to 273 μM in combination with 25uL of human plasma. However, no significant effect on drug inhibitionwas observed when substrate concentration was lowered over 273 μM to 65μM using 50 μL of plasma, which suggests drug dissociation is notpromoted by lower substrate levels with higher plasma volumes over thisrange.

Example 18 Design of Experiment Software

After identifying individual factors that contribute to the druginsensitivity of the original Auto PAF AH assay, JMP software (Design ofExperiment, herein “DOE”) was used to design experiments investigatinginteractions between individual factors and to identify optimalcombinations for detecting drug inhibition over an adequate dynamicrange.

DOE Experiment #1

The first DOE experiment focused on four factors including buffer R1volume (2 levels), plasma volume (4 levels), substrate concentration (8levels) and drug treatment (2 levels). [The indicated levels ofsubstrate concentration refer to the substrate concentration in thealiquot of premixed R2B/R1 added to each reaction unless otherwisenoted.] Although a full factorial combination of variables would require128 assay reactions, D-optimal design suggested 48 differentcombinations. These 48 reactions were performed in duplicate, using asingle plasma sample with or without prior in vitro incubation withFormula II at 37° C. for an hour. Substrate was directly diluted into R1and plasma was then added to start hydrolysis at room temperature. Vmaxand drug inhibition were calculated based on absorbance readings at 405nm over 5 minutes at room temperature.

JMP predicted that a combination of 15 μL or 25 μL of plasma and 110 μLof R1 containing 273 μM to 1100 μM substrate would indicate 90% orgreater drug inhibition. This set of conditions would also yieldreasonably high Vmax so that heavily drug-treated plasma would not fallbelow the lower limit of quantitation. The lowest substrateconcentration included in this experiment, 65 μM in 110 μL of R1, waspredicted to detect 93.41% drug inhibition when used with 25 μL ofplasma (final substrate concentration of 53 uM). However, Vmaxprediction was as low as 16.79 for the non-drug-treated sample. Suchcondition, although did not proceed for further optimization, could beused to assay specific sample sets that have low and narrow range ofLp-PLA2 activity.

DOE Experiment #2

The second DOE experiment focused on the conditions identified by theprior DOE experiment. It designed a full factorial combination of allvariables including R1 volume (1 level), plasma volume (2 levels),substrate concentration (4 levels) and drug treatment (4 levels).Thirty-two conditions were assayed in duplicate. The assay protocol wasidentical to the first DOE experiment. [The indicated levels ofsubstrate concentration again refer to the substrate concentration inthe aliquot of premixed R2B/R1 added to each reaction unless otherwisenoted.] Prediction Profiler predicted that 25 μL plasma and 110 μL of R1containing 545 μM substrate would generate a Vmax of 76 milliOD/min fornon-drug treated plasma and indicate close to 95% drug inhibition forplasma treated with 900 ng/mL of drug in vitro. Therefore, a modified,drug-sensitive assay uses 25 μL of plasma with 110 μL of R1 containing545 μM substrate for a final substrate concentration of 440 μM in theassay.

An alternative set of conditions was also identified (15 μL of plasmaand 110 μL of R1 containing 545 μM for a final substrate concentrationof 475 μM in the assay) that indicated 94% drug inhibition and Vmax=53milliOD/min for non-drug-treated plasma.

Example 19 Reaction Time

Four human plasma timepoint samples from a single subject, who wastreated in vivo with Lp-PLA2 inhibitor drug, were assayed for Lp-PLA2activity by a modified, drug-sensitive colorimetric assay containing 440μM substrate and 25 μL of plasma (described in Example 8). The same fourplasma samples were also assayed by the same assay protocol but with 50μL of plasma and 154 μM of substrate (see Table 17). The first 5 minutesof hydrolysis were monitored for each reaction and five Vmax values werecalculated based on time intervals of 1, 2, 3, 4 or 5 minutes from thestart of the reaction. Samples corresponding to high Lp-PLA2 inhibition(1 and 3 hour post-dose) exhibited higher Vmax values for longer assayreaction times when 25 μL plasma and 440 μM substrate were used. Thissuggests drug dissociation may occur under such condition wherecompetition between drug and substrate is relatively strong. Incontrast, Vmax values for 1 and 3 hour post-dose time points wereindependent of assay reaction time when more plasma and lower substratewas used (e.g., 50 μL plasma/154 μM substrate). However, Vmax valuestend to decrease with longer assay reaction times for samples with lowerdrug inhibition (0 and 0.5 hours) especially at higher plasma volume andlower substrate concentration as appreciable total substrate is consumedwith high Lp-PLA2 activity. Therefore, assay performance is affected byat least three factors affecting three attributes:

-   (1) High plasma volume, short incubation time and low substrate    concentration promote measurement of high levels of drug inhibition;-   (2) Low plasma volume, short incubation time and high substrate    concentration promote a high upper limit of quantitation;-   (3) High plasma volume, long incubation time and high substrate    concentration promote sensitive lower limits of quantitation.

The implementation of robotics is recommended to shorten the timebetween addition of substrate into plasma and the first absorbancereading on the plate reader. Current protocol assembles and mixes anentire microtiter plate of reactions and start plate reading 1 minuteafter starting the first reaction on the plate. Activity calculationsare based on data collected at 1 and 3 minutes in the microtiter platereader. However, since absorbance readings are collected for 10 minutesat 15-second intervals, depending on the objective of the assay andrange of activity seen with a specific sample set, shorter and/orearlier, or longer reaction time could be chosen to calculate Lp-PLA2activity.

TABLE 17 Effect of Reaction Time on Reaction Rate Under Different PlasmaVolume and Substrate Concentrations Vmax (milliOD/min) Time 25 μLPlasma/440 μM Substrate 50 μL Plasma/154 μM Substrate (Hr) 1 min 2 min 3min 4 min 5 min 1 min 2 min 3 min 4 min 5 min 0 125.20 118.65 115.01110.60 107.85 98.70 88.45 82.73 76.54 70.68 0.5 86.90 88.70 88.73 88.7988.38 76.50 72.85 69.05 64.78 61.71 1 19.00 21.05 23.37 25.72 27.9018.00 17.75 18.01 18.10 18.28 3 6.30 8.25 10.65 12.74 14.70 10.20 9.159.24 9.37 9.57

Example 20 Further Assay Testing

Inter-Assay Validation

Intra-assay variability was assessed using plasma samples from 10healthy (non-fasted) human subjects. Six replicates of plasma from eachsubject were assayed on the same assay plate. The CV for individualsubjects ranged from 2.57 to 9.14% with an average intra-assay CV of5.36% as shown in Table 18.

TABLE 18 Intra-Assay Validation Lp-PLA2 Activity (nmol/min/mL) ReplicateSubject No. No. #6954966 #5149192 #6839829 #5147931 #5181480 #5149190#5149188 #6954955 #6955001 #6716001 1 112.26 64.53 147.94 138.05 94.56105.37 132.26 140.35 140.56 139.30 2 115.68 78.40 137.14 135.75 93.5997.35 109.41 135.33 124.53 145.92 3 108.92 74.29 156.52 146.62 90.45113.87 111.78 129.55 119.30 145.30 4 113.24 74.49 174.22 143.55 93.10102.86 107.25 138.82 116.93 140.14 5 108.01 69.62 138.47 140.28 92.82120.07 107.87 128.85 130.31 140.14 6 113.31 80.14 146.83 145.16 91.85114.70 112.89 134.70 136.59 147.87 Average 111.90 73.58 150.19 141.5792.73 109.04 113.58 134.60 128.04 143.11 % CV 2.60 7.81 9.14 3.00 1.547.84 8.29 3.48 7.38 2.57Inter-Assay Variability

Inter-assay variability was assessed using plasma samples from 10healthy human subjects (non-fasted), assayed in three separate assays ondifferent days. The inter-assay CV for individual plasma samples rangedfrom 1.90 to 23.78% with an average inter-assay CV of 7.59%. Plasma fromSubject #5181480 (inter-assay CV=23.78%) had a white/turbid appearanceafter brief centrifugation, suggesting high lipid content in the sampleas shown in Table 19.

TABLE 19 Inter-Assay Variability Lp-PLA2 Activity (nmol/min/mL) AssaySubject No. No. #6954966 #5149192 #6839829 #5147931 #5181480 #5149190#5149188 #6954955 #6955001 #6716001 1 111.90 73.58 150.19 141.57 92.73109.04 113.58 134.60 128.04 143.11 2 105.75 76.77 143.36 127.71 58.21106.36 112.86 106.26 116.87 134.58 3 117.70 83.78 147.66 118.56 70.1797.80 116.94 120.69 130.48 134.02 Average 111.79 78.04 147.07 129.2873.70 104.40 114.46 120.52 125.13 137.24 % CV 5.34 6.69 2.35 8.96 23.785.62 1.90 11.76 5.80 3.71Inter-Operator Variability

Inter-operator variability was assessed using plasma samples from 10healthy subjects assayed by three different operators on different days.The inter-operator CV for individual plasma samples ranged from 5.11 to14.91% with an average inter-operator CV of 8.32% as shown in Table 20.

TABLE 20 Inter-Operator Variability Lp-PLA2 Activity (nmol/min/mL)Operator Subject No. No. #6954966 #5149192 #6839829 #5147931 #5181480#5149190 #5149188 #6954955 #6955001 #6716001 1 111.79 78.04 147.07129.28 73.70 104.40 114.46 120.52 125.13 137.24 2 107.20 70.46 128.55128.06 66.74 100.98 106.22 114.00 114.09 109.69 3 98.60 74.85 136.87111.37 62.05 82.44 102.25 100.23 112.41 104.82 Average 105.86 74.45137.50 122.90 67.50 95.94 107.64 111.58 117.21 117.25 % CV 6.32 5.116.75 8.14 8.69 12.31 5.79 9.28 5.89 14.91Freeze/Thaw Effect

Plasma samples are normally received and stored frozen. In the case ofrepeat analysis, samples are commonly subject to freeze/thaw cycles. Tenplasma samples were analyzed after each of four freeze/thaw cycles. Nodefinitive trend in Lp-PLA2 values was observed, indicating samples maybe frozen and thawed four times, a shown in Table 20.

TABLE 21 Freeze/Thaw Effect Lp-PLA2 Activity (nmol/min/mL) Freeze/Subject No. Thaw 6954966 5149192 6839829 5147931 5181480 5149190 51491886954955 6955001 6716001 1 111.90 73.58 150.19 141.57 92.73 109.04 113.58134.60 128.04 143.11 2 105.75 76.77 143.36 127.71 58.21 106.36 112.86106.26 116.87 134.58 3 112.72 82.20 141.72 107.84 53.78 81.48 116.7099.18 130.55 125.98 4 101.09 68.94 105.00 95.96 55.96 97.12 98.05 122.05105.40 169.87 Average 107.87 75.37 135.07 118.27 65.17 98.50 110.30115.52 120.21 143.38 % CV 5.08 7.39 15.09 17.18 28.33 12.63 7.56 13.779.59 13.24

Example 21 Higher Drug Inhibition and Assay Dynamic Range

Four hundred forty microMolar (440 μM) substrate and 25 μL of plasmasample volume were selected for use in the current modified assayprotocol since they offered high detectable in vivo drug inhibitionwhile maintaining adequate assay dynamic range. However, furtherlowering substrate concentration and/or increasing plasma sample volumein the assay could detect higher measurable drug inhibition in vivo atexpense of assay dynamic range. Plasma samples from 5 human subjectsreceiving Formula II for 9 days in a clinical study were collected onday 10 at different time points. Pre-dose plasma samples for eachsubject on day 0 of the study were also available. When 440 μM substrateand 25 μL of plasma were used in the assay, maximal 68% drug inhibitionwas observed in Subject N030 at 4 hour-timepoint as shown in Table 22.Lowering substrate concentration to 112 μM while maintaining 25 μl ofplasma volume increased drug inhibition to 76% at this time point.Further increase in drug inhibition to 79% at 4 hour-timepoint, wasachieved with both lower substrate concentration of 112 μM and higherplasma volume of 45 μL.

TABLE 22 Improved Detectable Inhibition in Human Subjects AdministeredLp-PLA2 Inhibitor Lp-PLA activity (nmol/min/mL) % Inhibition Time(hr)440 μM/25 μL 112 μM/25 μL 112 μM/45 μL 440 μM/25 μL 112 μM/25 μL 112μM/45 μL Pre-dose 110.59 38.95 35.59 0 0 0 0 51.85 14.41 11.58 53 63 670.5 48.74 15.08 8.87 56 61 75 1 47.39 12.10 9.55 57 69 73 2 41.97 14.5411.48 62 63 68 3 40.67 11.51 6.54 63 70 82 4 35.88 9.37 7.31 68 76 79 643.32 9.62 6.12 61 75 83 9 39.37 10.59 7.52 64 73 79 12 39.66 11.22 8.8564 71 75 18 48.87 13.53 11.32 56 65 68 24 39.08 8.24 6.30 65 79 82

Plasma samples from the other four subjects were analyzed using both 440μM substrate/25 μL plasma and 112 μM substrate/45 μL plasma assaycondition. The maximal drug inhibition detected with 440 μM substrateand 25 μL plasma was between 68% and 80% in these subjects as shown inTable 23. However, the use of 112 μM substrate and 45 μL of plasmafurther improved measurable drug inhibition in the same subjects withmaximal inhibition between 87% and 98%. With 112 μM substrate/45 μLplasma, the absolute Lp-PLA2 activity value decreased significantly withthose at highest drug inhibition points approaching lower limit ofp-nitrophenol linear detection range described in Example 12. Forexample, the 4 hour-timepoint plasma for Subject N028 showed Lp-PLAactivity of 1.31 nmol/min/mL (see Table 23). Such level of activitywould only generate 0.12 nmol of p-nitrophenol in two minutes of assaytime based on a modified drug sensitive colorimetric assay described inExample 8, slightly above the low end of p-nitrophenol linear detectionrange 0.1 nmol.

TABLE 23 Improved Detectable Inhibition Lp-PLA2 Activity (nmol/min/mL) %Inhibition 440 μM/ 112 μM/ 440 μM/ 112 μM/ Subject No. Time (hr) 25 μL45 μL 25 μL 45 μL N008 Pre-dose 104.20 25.81 0 0 0 47.06 12.65 55 51 0.545.87 12.67 47 51 1 53.40 11.37 49 56 2 61.13 13.14 41 49 3 52.48 13.4250 48 4 46.51 6.19 55 76 6 33.95 3.27 67 87 9 43.87 6.21 58 76 12 33.665.74 68 78 18 62.77 6.54 40 75 24 65.38 11.16 37 57 N009 Pre-dose 168.2842.62 0 0 0 69.03 14.49 59 66 0.5 66.97 16.97 60 60 1 69.71 19.42 59 542 73.99 17.16 56 60 3 68.87 13.12 59 69 4 54.75 3.64 67 91 6 53.19 10.9768 74 9 60.76 11.27 64 74 12 76.09 12.58 55 70 18 71.39 18.51 58 57 2469.12 16.69 59 61 N028 Pre-dose 188.82 53.36 0 0 0 60.50 7.80 68 85 0.554.96 3.29 71 94 1 53.70 6.28 72 88 2 62.98 7.24 67 86 3 64.87 4.06 6692 4 42.65 1.31 77 98 6 37.35 3.55 80 93 9 43.99 2.99 77 94 12 45.885.30 76 90 18 53.56 13.40 72 75 24 56.68 11.69 70 78 N029 Pre-dose139.03 73.80 0 0 0 70.50 16.59 49 78 0.5 65.92 15.45 53 79 1 73.03 16.7647 77 2 62.98 15.33 55 79 3 46.81 5.88 66 92 4 46.09 5.39 67 83 6 41.094.11 70 94 9 44.12 6.54 68 91 12 50.13 8.64 64 88 18 59.08 10.71 58 8524 62.56 19.75 55 73

To define the assay dynamic range for using 112 μM of substrate and 45μl of plasma, serially diluted recombinant human Lp-PLA2 protein wasassayed for Lp-PLA2 activity. The activity of hrLp-PLA2 between 4.88 to312.50 ng/mL demonstrated linearity with an R value of 0.96 (see Table24). Therefore, the dynamic range appears to be between 2.71 and 84.14nmol/min/mL. Compared to the dynamic range between 4.4 and 397nmol/min/mL for 440 μM of substrate and 25 μL of plasma determined inExample 12 using hrLp-PLA, 112 μM substrate/45 μL plasma, althoughlowering low limit of quantitation, could only offer limited assayrange. Therefore, such conditions with lower substrate concentration andhigher sample volume could be used when higher measurable in vivoinhibition is desired while the range of Lp-PLA2 activity for testsamples is limited or could be compromised. One example such conditionscould be applied to is in clinical studies for Lp-PLA2 inhibitor drugsin which most post-drug test samples demonstrate low Lp-PLA2 activityresulted from drug inhibition. Earlier and shorter reaction time couldbe considered to use in activity calculation to improve assay dynamicrange when such conditions are used.

TABLE 24 Assay of Recombinant Human Lp-PLA2 Using 112 μM Substrate/45 μLPlasma hrLp-PLA2 (ng/mL) Activity (nmol/min/mL) 0.00 0.00 1.22 0.18 2.440.62 4.88 2.71 9.77 5.13 19.53 9.27 39.06 17.81 78.13 30.16 156.25 51.62312.50 84.14 625.00 109.44 1250.00 93.90

The assay dynamic range of 2.71-84.14 nmol/min/mL determined byhrLp-PLA2 was calculated based on absorbance change between 3 minutesand 1 minute after the start of reaction. When absorbance differencesbetween 1 minute and 0 minutes of the reaction were used to calculateLp-PLA2 activity from the same data, dynamic range was significantlyimproved to 3.2-196.5 nmol/min/mL (see Table 25). Shortening reactiontime to 30 seconds in activity calculation showed little furtherimprovement.

TABLE 25 Effect of Reaction Time Used for Activity Calculation on AssayDynamic Range Reaction Time Used for hrLp-PLA2 Activity ActivityCalculation (ng/ml) (nmol/min/mL) R 3 min-1 min 14.6-938  2.71-84.1 0.98 1 min-0   14.6-3750  3.2-196.5 0.97 30 sec-0   14.6-3750 3.4-210 0.98

Example 22 Detection of Lp-PLA2 Activity and its In Vitro DrugInhibition in Serum

To assess the utility of a modified colorimetric assay for measuringLp-PLA2 activity and particularly its drug inhibition in serum, 10 serumsamples collected from normal donors were assayed. The measured Lp-PLA2activity shown in Table 26 ranged between 130 and 190 nmol/min/mL forthese serum samples. The % CV between duplicates of each sample wasmostly less than 5%. No matched plasma samples were available foranalysis. However, pre-dose plasma samples from 14 subjects described inExample 10 and 11 showed a comparable range of Lp-PLA2 activity between80 and 200 nmol/min/mL.

TABLE 26 Lp-PLA2 Activity in Ten Serum Samples Sample Activity(nmol/min/mL) % Inhibition BRH28858 145.57 5.28 BRH28859 131.36 1.73BRH28860 177.74 0.75 BRH28861 187.11 0.79 BRH28862 155.68 1.65 BRH28865144.04 3.28 BRH28866 133.28 1.52 BRH28867 131.28 2.92 BRH28868 137.351.15 BRH28869 140.00 3.66

Since no serum samples were available from human subjects administeredLp-PLA2 inhibitors, 2 serum samples, BRH28861 and BRH28867, werepre-treated in vitro with different doses of Lp-PLA2 inhibitor FormulaII. The dose range used in vitro contained the range of in vivo plasmaconcentrations of such inhibitor in human subjects receiving drug duringclinical studies of Formula II. Based on pharmacokinetics data, 90 ng/mLrepresented the peak plasma level of Formula II when administered invivo. These in vitro drug-treated serum samples were then assayed forLp-PLA2 activity by a modified colorimetric assay. Table 27 showed thatBRH28861 and BRH28867 reached 88.25% and 90.77% inhibition of Lp-PLA2activity, respectively, when treated with 90 ng/mL of Formula II invitro. Higher drug dose at 900 ng/mL level further increased druginhibition to 97.71% and 92.28% respectively in these two serum samples.

TABLE 27 In vitro Drug Inhibition of Serum Lp-PLA2 Activity DrugActivity (nmol/min/mL) % Inhibition (ng/mL) BRH28861 BRH28867 BRH28861BRH28867 0 153.90 110.17 0.00 0.00 1 166.90 103.62 −8.44 5.95 2 137.4692.54 10.69 16.00 5 154.60 103.66 −0.45 5.91 10 137.70 82.47 10.53 25.1430 55.54 40.38 63.91 63.35 60 16.34 13.66 89.38 87.60 90 18.08 10.1788.25 90.77 900 3.52 7.91 97.71 92.82

All publications and references, including but not limited to patentsand patent applications, cited in this specification are hereinincorporated by reference in their entirety as if each individualpublication or reference were specifically and individually indicated tobe incorporated by reference herein as being fully set forth. Any patentapplication to which this application claims priority is alsoincorporated by reference herein in its entirety in the manner describedabove for publications and references.

What is claimed is:
 1. An Lp-PLA2 colorimetric activity assay mixturethat can detect Lp-PLA2 activity in the presence of an inhibitor ofLp-PLA2 activity, the assay mixture made by combining: a substratesolution comprising1-myristoyl-2-(p-nitrophenylsuccinyl)phosphatidylcholine; a buffersolution comprising a detergent; and a plasma sample comprising aninhibitor of Lp-PLA2, wherein the plasma sample has been diluted about 3to 9 fold in the assay mixture, and wherein the concentration ofsubstrate in the assay mixture is less than or equal to about the Km ofthe substrate.
 2. The assay mixture of claim 1, wherein the buffersolution comprises HEPES, and EDTA and has a pH of 7.6.
 3. The assaymixture of claim 1, wherein the buffer solution comprises CHAPS and1-nonane-sulfonate.
 4. The assay mixture of claim 1, wherein the buffersolution and substrate solution do not contain citric acid.
 5. AnLp-PLA2 colorimetric activity assay mixture that can detect Lp-PLA2activity in the presence of an inhibitor of Lp-PLA2 activity, the assaymixture made by combining: a substrate solution comprising1-myristoyl-2-(p-nitrophenylsuccinyl)phosphatidylcholine, a buffersolution comprising a detergent, and a plasma sample comprising aninhibitor of Lp-PLA2, wherein the plasma sample has been diluted about 3to 9 fold in the assay mixture, and wherein the concentration ofsubstrate is between about 53 μM and about 1125 μM in the assay mixture.6. The assay mixture of claim 5, wherein the buffer solution comprisesHEPES, and EDTA and has a pH of 7.6.
 7. The assay mixture of claim 5,wherein the buffer solution comprises CHAPS and 1-nonane-sulfonate. 8.The assay mixture of claim 5, wherein the concentration of substrate inthe assay mixture is between about 53 μM and about 200 μM.
 9. The assaymixture of claim 5, wherein the concentration of substrate in the assaymixture is between about 53 μM and about 440 μM.
 10. The assay mixtureof claim 5, wherein the concentration of substrate in the assay mixtureis about 112 μM.
 11. The assay mixture of claim 5, wherein the assaymixture is contained in a multiwell plate.
 12. The assay mixture ofclaim 5, wherein the assay mixture is maintained at room temperature.13. The assay mixture of claim 5, wherein the buffer solution andsubstrate solution do not contain citric acid.
 14. A colorimetric methodof determining inhibition of Lp-PLA2 enzyme activity in a sample ofblood plasma obtained from a human patient taking an inhibitor ofLp-PLA2, the method comprising: forming an assay mixture by combining aplasma sample with a buffer solution comprising a detergent and asubstrate solution comprising1-myristoyl-2-(p-nitrophenylsuccinyl)phosphatidylcholine so that theplasma sample is diluted about 3 to about 9 fold and the concentrationof substrate in the assay mixture is less than or equal to the Km of thesubstrate; and colorimetrically sampling the assay mixture to determineactivity of the Lp-PLA2 enzyme.
 15. The method of claim 14, wherein themethod is performed without preincubating a dilution of the plasmasample prior to the addition of the substrate solution.
 16. The methodof claim 14, wherein the concentration of substrate in the assay mixtureis equal to about the Km of the substrate.
 17. The method of claim 14,wherein colorimetrically sampling is performed at 405 nm.
 18. The methodof claim 14, wherein colorimetrically sampling is performed at roomtemperature.
 19. The method of claim 14, wherein colorimetricallysampling the assay mixture is performed so that a first absorbancereading is taken within a minute of forming the assay mixture.
 20. Themethod of claim 14, wherein the buffer solution and substrate solutiondo not contain citric acid.
 21. A colorimetric method of determininginhibition of Lp-PLA2 enzyme activity in a sample of blood plasmaobtained from a human patient taking an inhibitor of Lp-PLA2, the methodcomprising: forming an assay mixture by combining a plasma sample from apatient with a buffer solution comprising a detergent and a substratesolution comprising1-myristoyl-2-(p-nitrophenylsuccinyl)phosphatidylcholine so that theplasma sample is diluted about 3 to about 9 fold and the concentrationof substrate is between about 53 μM to about 1125 μM in the assaymixture; and colorimetrically sampling to determine an absorbance fromthe colorimetric sample solution.
 22. The method of claim 21, furthercomprising preparing colorimetric standards at various concentrationsand generating a standard curve from the standards.
 23. The method ofclaim 21, wherein the concentration of substrate in the assay mixture isbetween about 53 μM and about 200 μM.
 24. The method of claim 21,wherein the concentration of substrate in the assay mixture is betweenabout 53 μM and about 440 μM.
 25. The method of claim 21, wherein theconcentration of substrate in the assay mixture is about 112 μM.
 26. Themethod of claim 21, wherein colorimetrically sampling is performed atroom temperature.
 27. The method of claim 21, wherein colorimetricallysampling the assay mixture is performed so that a first absorbancereading is taken within a minute of forming the assay mixture.
 28. Themethod of claim 21, wherein the method is performed withoutpreincubating a dilution of the plasma sample prior to the addition ofthe substrate solution.
 29. The method of claim 21, wherein the buffersolution and substrate solution do not contain citric acid.